Single variable domains against the notch pathways

ABSTRACT

The present invention relates to amino acid sequences (also referred to herein as “single variable domain(s) of the invention”. “Nanobody™ of the invention” or “Nanobodies™ of the invention”, ilpolypeptide(s) of the invention”) that are directed against (as defined herein) members of the Notch signalling pathway such as the four mammalian members of the Notch Receptors (Notch-1, Notch-2, Notch-3, Notch-4, hereinafter also “Notch Receptors”) and the five mammalian members of the Notch Ligands (the Delta-like family with “DLL1”, “DLL3”, “DLL4” wherein “DLL” stands for Delta-like ligand; the Jagged family with “Jagged-1”, “Jagged-2”, hereinafter also “Notch Ligands”), as well as to constructs that comprise or essentially consist of one or more such single variable domain(s) (also referred to herein as “construct of the invention” and “constructs of the invention”, “Nanobody™ of the invention” or “Nanobodies™ of the invention”, respectively). In particular, this invention relates to Notch pathway interfering single variable domains down- or up-regulating Notch signaling (also referred to herein “Notch-antagonists” or “Notch-agonists”).

The present invention relates to amino acid sequences (also referred toherein as “single variable domain(s) of the invention”, “Nanobody™ ofthe invention” or “Nanobodies™ of the invention”, “polypeptide(s) of theinvention”) that are directed against (as defined herein) members of theNotch signalling pathway such as the four mammalian members of the NotchReceptors (Notch-1, Notch-2, Notch-3, Notch-4, hereinafter also “NotchReceptors”) and the five mammalian members of the Notch Ligands (theDelta-like family with “DLL1”, “DLL3”, “DLL4” wherein “DLL” stands forDelta-like ligand; the Jagged family with “Jagged-1”, “Jagged-2”,hereinafter also “Notch Ligands”), as well as to constructs thatcomprise or essentially consist of one or more such single variabledomain(s) (also referred to herein as “construct of the invention” and“constructs of the invention”, “Nanobody™ of the invention” or“Nanobodies™ of the invention”, respectively). In particular, thisinvention relates to Notch pathway interfering single variable domainsdown- or up-regulating Notch signaling (also referred to herein as“Notch-antagonists” or “Notch-agonists”).

The invention also relates to nucleic acids encoding such singlevariable domains or constructs (also referred to herein as “nucleicacids of the invention”, “nucleic acid of the invention”, “nucleotidesequence of the invention” or “nucleotide sequences of the invention”);to methods for preparing such single variable domains or constructs; tohost cells expressing or capable of expressing such single variabledomains or constructs; to compositions, and in particular topharmaceutical compositions, that comprise such single variable domainsor constructs; and to uses of such single variable domains, constructs,nucleic acids, host cells and/or compositions, in particular forprophylactic, therapeutic or diagnostic purposes, such as the uses incancer, immunomodulation and neurodegenerative disorders and inparticular the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

The Notch pathway is known to control cell fate choices in many tissuesduring development and differentiation. As for cancer, numerousobservations suggest that chronically active Notch signalling isassociated with the transformed phenotype in many cells. Overexpressionof apparently full-length Notch receptors and ligands is observed inseveral common epithelial malignancies and tumor cell lines. In othercases, activated Notch signalling results from deletions that produceconstitutively active Notch receptors (Miele et al., supra). Examples ofpossibly suitable uses of a polypeptide of the invention in cancer aremainly in the form of Notch-antagonists and are for example: cervicalcarcinomas, pre-neoplastic lesions in cervical epithelium, endometrialcarcinomas, T-cell acute lymphoblastic leukaemia (T-ALL), acutepromyelocytic leukaemia, erythroleukemia, glioblastoma multiforme,neuroblastoma and medulloblastoma to pleural mesothelioma (Miele et al,supra), disease with excessive angiogenesis (Thurston G. et. Al, 2007,Nature 7, 327-331). Blockade of DLL4 inhibits tumour growth by promotingnon-productive angiogenesis (WO2007070671).

As for neurodegenerative disorders, Notch signalling has been known formany years to play a pivotal role in the development of the centralnervous system and, more recently also in neuropathology. Examples ofpossibly suitable uses of a polypeptide of the invention inneurodegenerative diseases are mainly in the form of Notch-agonists andare for example: CADASIL, Alzheimer's disease, familial Alzheimer'sdisease, demyelinating disorders such as Multiple Sclerosis,neurological lesions such as spinal cord injuries, stroke (Miele et al.,supra).

As for immunomodulation, considerable evidence points to multiple rolesof Notch signalling in the immune system. It is well established thatNotch signalling plays a role in T-cell maturation, in particular byinhibiting the “differentiation” program that is triggered afteractivation of T-cells by a cognate interaction. Hence, Notch-agonistsmay be used for immunosuppressive or tolerogenic interferences (uses intransplantation/graft versus host diseases); Notch-antagonists mayamplify an immune response.

The single variable domains and constructs of the present invention cangenerally be used to modulate, and in particular inhibit and/or prevent,binding of Notch Ligands to Notch Receptors, and thus to modulate, andin particular inhibit or prevent, the signalling that is mediated byNotch Ligands and/or Notch Receptors, to modulate the biologicalpathways in which Notch Ligands and/or Notch Receptors are involved,and/or to modulate the biological mechanisms, responses and effectsassociated with such signalling or these pathways.

As such, the single variable domains and constructs of the presentinvention can be used for the prevention and treatment (as definedherein) of cancer, neurodegenerative diseases and immunomodulatorydiseases. Generally, cancer, neurodegenerative diseases andimmunomodulatory diseases can be defined as diseases and disorders thatcan be prevented and/or treated, respectively, by suitably administeringto a subject in need thereof (i.e. having the disease or disorder or atleast one symptom thereof and/or at risk of attracting or developing thedisease or disorder) of either a polypeptide or composition of theinvention (and in particular, of a pharmaceutically active amountthereof) and/or of a known active principle active against members ofthe Notch signalling pathway members of the Notch signalling pathway ora biological pathway or mechanism in which members of the Notchsignalling pathway is involved (and in particular, of a pharmaceuticallyactive amount thereof). Examples of such cancer, neurodegenerativediseases and immunomodulatory diseases will be clear to the skilledperson based on the disclosure herein, and for example include thefollowing diseases and disorders: Alzheimer, cervical carcinomas,pre-neoplastic lesions in cervical epithelium, endometrial carcinomas,T-cell acute lymphoblastic leukaemia (T-ALL), acute promyelocyticleukaemia, erythroleukemia, glioblastoma multiforme, neuroblastoma andmedulloblastoma to pleural mesothelioma (Miele et al, supra), diseasewith excessive angiogenesis (Thurston G. et. Al, 2007, Nature 7,327-331), CADASIL, Alzheimer's disease, familial Alzheimer's disease,demyelinating disorders such as Multiple Sclerosis, neurological lesionssuch as spinal cord injuries, stroke (Miele et al., supra),transplantation/graft versus host diseases.

In particular, the single variable domains and constructs of the presentinvention can be used for the prevention and treatment of cancer,neurodegenerative diseases and immunomodulatory diseases which arecharacterized by excessive and/or unwanted signaling mediated by membersof the Notch signalling pathway or by the pathway(s) in which members ofthe Notch signalling pathway are involved. Examples of such cancer,neurodegenerative diseases and immunomodulatory diseases will again beclear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, the single variable domains of theinvention can for example be used to prevent and/or to treat alldiseases and disorders that are currently being prevented or treatedwith active principles that can modulate Notch pathway-mediatedsignalling, such as those mentioned in the prior art cited above. It isalso envisaged that the single variable domains of the invention can beused to prevent and/or to treat all diseases and disorders for whichtreatment with such active principles is currently being developed, hasbeen proposed, or will be proposed or developed in future. In addition,it is envisaged that, because of their favourable properties as furtherdescribed herein, the single variable domains of the present inventionmay be used for the prevention and treatment of other diseases anddisorders than those for which these known active principles are beingused or will be proposed or developed; and/or that the single variabledomains of the present invention may provide new methods and regimensfor treating the diseases and disorders described herein.

Other applications and uses of the single variable domains of theinvention will become clear to the skilled person from the furtherdisclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as constructs comprising the same, that can beused in the diagnosis, prevention and/or treatment of cancer,neurodegenerative diseases and immunomodulatory diseases and of thefurther diseases and disorders mentioned herein; and to provide methodsfor the diagnosis, prevention and/or treatment of such diseases anddisorders that involve the administration and/or use of such agents andconstructs.

In particular, it is an object of the invention to provide suchpharmacologically active agents, constructs and/or methods that havecertain advantages compared to the agents, constructs and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as constructs comprising the same, for the diagnosis,prevention and/or treatment of cancer, neurodegenerative diseases andimmunomodulatory diseases and of the further diseases and disordersmentioned herein; and to provide methods for the diagnosis, preventionand/or treatment of such diseases and disorders that involve theadministration and/or the use of such therapeutic proteins andconstructs.

Accordingly, it is a specific object of the present invention to providesingle variable domains that are directed against (as defined herein)members of the Notch signalling pathway, in particular against membersof the Notch signalling pathway from a warm-blooded animal, more inparticular against members of the Notch signalling pathway from amammal, and especially against human members of the Notch signallingpathway, and to provide single variable domains comprising oressentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such single variable domains that are suitable for prophylactic,therapeutic and/or diagnostic use in a warm-blooded animal, and inparticular in a mammal, and more in particular in a human being.

More in particular, it is a specific object of the present invention toprovide such single variable domains that can be used for theprevention, treatment, alleviation and/or diagnosis of one or morediseases, disorders or conditions associated with members of the Notchsignalling pathway and/or mediated by members of the Notch signallingpathway (such as the diseases, disorders and conditions mentionedherein) in a warm-blooded animal, in particular in a mammal, and more inparticular in a human being.

It is also a specific object of the invention to provide such singlevariable domains that can be used in the preparation of pharmaceuticalor veterinary compositions for the prevention and/or treatment of one ormore diseases, disorders or conditions associated with and/or mediatedby members of the Notch signalling pathway (such as the diseases,disorders and conditions mentioned herein) in a warm-blooded animal, inparticular in a mammal, and more in particular in a human being.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, single variable domains andconstructs that are described herein.

In general, the invention provides single variable domains that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to members of the Notch signalling pathway; as well ascompounds and constructs, and in particular proteins and single variabledomains, that comprise at least one such amino acid sequence.

More in particular, the invention provides single variable domains thatcan bind to members of the Notch signalling pathway with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A) value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein; as well as compounds and constructs,and in particular proteins and single variable domains, that comprise atleast one such amino acid sequence.

In particular, single variable domains of the invention are preferablysuch that they:

-   -   bind to members of the Notch signalling pathway with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to members of the Notch signalling pathway with a        k_(on)-rate of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,        preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably        between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹        and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to members of the Notch signalling pathway with a k_(off)        rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a        near irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to members of the Notch signallingpathway with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM.

Some preferred IC50 values for binding of the single variable domains orsingle variable domains of the invention to members of the Notchsignalling pathway will become clear from the further description andexamples herein.

For binding to members of the Notch signalling pathway, an amino acidsequence of the invention will usually contain within its amino acidsequence one or more amino acid residues or one or more stretches ofamino acid residues (i.e. with each “stretch” comprising two or aminoacid residues that are adjacent to each other or in close proximity toeach other, i.e. in the primary or tertiary structure of the amino acidsequence) via which the single variable domains of the invention canbind to members of the Notch signalling pathway, which amino acidresidues or stretches of amino acid residues thus form the “site” forbinding to members of the Notch signalling pathway (also referred toherein as the “antigen binding site”).

The single variable domains provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more single variable domains of theinvention and which may optionally further comprise one or more furthersingle variable domains (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more singlevariable domains of the invention may be used as a binding unit in sucha protein or polypeptide, which may optionally contain one or morefurther single variable domains that can serve as a binding unit (i.e.against one or more other targets than members of the Notch signallingpathway), so as to provide a monovalent, multivalent or multispecificpolypeptide of the invention, respectively, all as described herein.Such a protein or polypeptide may also be in essentially isolated form(as defined herein).

The single variable domains of the invention as such preferablyessentially consist of a single amino acid chain that is not linked viadisulphide bridges to any other amino acid sequence or chain (but thatmay or may not contain one or more intramolecular disulphide bridges.For example, it is known that Nanobodies—as described herein—maysometimes contain a disulphide bridge between CDR3 and CDR1 or FR2).However, it should be noted that one or more single variable domains ofthe invention may be linked to each other and/or to other singlevariable domains (e.g. via disulphide bridges) to provide peptideconstructs that may also be useful in the invention (for example Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs. Reference is for example made to the review byHolliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the single variable domains of the invention (as well as compounds,constructs and single variable domains comprising the same) arepreferably directed against human members of the Notch signallingpathway; whereas for veterinary purposes, the single variable domains ofthe invention are preferably directed against members of the Notchsignalling pathway from the species to be treated, or are at leastcross-reactive with members of the Notch signalling pathway from thespecies to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against membersof the Notch signalling pathway, contain one or more further bindingsites for binding against other antigens, proteins or targets.

The efficacy of the single variable domains of the invention, and ofconstructs comprising the same, can be tested using any suitable invitro assay, cell-based assay, in vivo assay and/or animal model knownper se, or any combination thereof, depending on the specific disease ordisorder involved. Suitable assays and animal models will be clear tothe skilled person, and for example include mouse models for variousdiseases where a member of the Notch signalling pathway is involved havebeen developed (T. Gridley, Human Molecular Genetics, 2003, Vol., 12,Review Issue 1, R9-R13), as well as the assays and animal models used inthe experimental part below and in the prior art cited herein.

Also, according to the invention, single variable domains that aredirected against members of the Notch signalling pathway from a firstspecies of warm-blooded animal may or may not show cross-reactivity withmembers of the Notch signalling pathway from one or more other speciesof warm-blooded animal. For example, single variable domains directedagainst human members of the Notch signalling pathway may or may notshow cross reactivity with members of the Notch signalling pathway fromone or more other species of primates (such as, without limitation,monkeys from the genus Macaca (such as, and in particular, cynomolgusmonkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta))and baboon (Papio ursinus)) and/or with members of the Notch signallingpathway from one or more species of animals that are often used inanimal models for diseases (for example mouse, rat, rabbit, pig or dog),and in particular in animal models for diseases and disorders associatedwith members of the Notch signalling pathway (such as the species andanimal models mentioned herein). In this respect, it will be clear tothe skilled person that such cross-reactivity, when present, may haveadvantages from a drug development point of view, since it allows thesingle variable domains against human members of the Notch signallingpathway to be tested in such disease models.

More generally, single variable domains of the invention that arecross-reactive with members of the Notch signalling pathway frommultiple species of mammal will usually be advantageous for use inveterinary applications, since it will allow the same amino acidsequence or polypeptide to be used across multiple species. Thus, it isalso encompassed within the scope of the invention that single variabledomains directed against members of the Notch signalling pathway fromone species of animal (such as single variable domains against humanmembers of the Notch signalling pathway) can be used in the treatment ofanother species of animal, as long as the use of the/or single variabledomains provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of members ofthe Notch signalling pathway against which the single variable domainsof the invention are directed. For example, the single variable domainsmay or may not be directed against an “interaction site” (as definedherein). However, it is generally assumed and preferred that the singlevariable domains of the invention are preferably directed against aninteraction site (as defined herein), and in particular against at leastpartly either the ligand site or the receptor site of theligand-receptor interaction sites). Thus, in one preferred, butnon-limiting aspect, the single variable domains of the invention aredirected against ligand-receptor interaction site of the Notch Ligands(supra) or/and are directed against the ligand-receptor interaction siteof the Notch Receptors (supra), and are as further defined herein.

As further described herein, a polypeptide of the invention may containtwo or more single variable domains of the invention that are directedagainst members of the Notch signaling pathway. Generally, such singlevariable domains will bind to members of the Notch signalling pathwaywith increased avidity compared to a single amino acid sequence of theinvention. Such a polypeptide may for example comprise two singlevariable domains of the invention that are directed against the sameantigenic determinant, epitope, part, domain, subunit or confirmation(where applicable) of members of the Notch signalling pathway (which mayor may not be an interaction site); or comprise at least one “first”amino acid sequence of the invention that is directed against a firstsame antigenic determinant, epitope, part, domain, subunit orconfirmation (where applicable) of members of the Notch signallingpathway (which may or may not be an interaction site); and at least one“second” amino acid sequence of the invention that is directed against asecond antigenic determinant, epitope, part, domain, subunit orconfirmation (where applicable) different from the first (and whichagain may or may not be an interaction site). Preferably, in such“biparatopic” single variable domains of the invention, at least oneamino acid sequence of the invention is directed against an interactionsite (as defined herein), although the invention in its broadest senseis not limited thereto.

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the single variable domains may be suchthat they compete with the cognate binding partner (e.g. the ligand,receptor or other binding partner, as applicable) for binding to thetarget, and/or such that they (fully or partially) neutralize binding ofthe binding partner to the target.

It is also within the scope of the invention that, where applicable, anamino acid sequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations ofmembers of the Notch signalling pathway. In such a case, the antigenicdeterminants, epitopes, parts, domains or subunits of members of theNotch signalling pathway to which the single variable domains of theinvention bind may be essentially the same (for example, if members ofthe Notch signalling pathway contains repeated structural motifs oroccurs in a multimeric form) or may be different (and in the lattercase, the single variable domains of the invention may bind to suchdifferent antigenic determinants, epitopes, parts, domains, subunits ofmembers of the Notch signalling pathway with an affinity and/orspecificity which may be the same or different). Also, for example, whenmembers of the Notch signalling pathway exists in an activatedconformation and in an inactive conformation, the single variabledomains of the invention may bind to either one of these confirmation,or may bind to both these confirmations (i.e. with an affinity and/orspecificity which may be the same or different). Also, for example, thesingle variable domains of the invention may bind to a conformation ofmembers of the Notch signalling pathway in which it is bound to apertinent ligand, may bind to a conformation of members of the Notchsignalling pathway in which it not bound to a pertinent ligand, or maybind to both such conformations (again with an affinity and/orspecificity which may be the same or different).

It is also expected that the single variable domains of the inventionwill generally bind to all naturally occurring or synthetic analogs,variants, mutants, alleles, parts and fragments of the members of theNotch signalling pathway; or at least to those analogs, variants,mutants, alleles, parts and fragments of members of the Notch signallingpathway that contain one or more antigenic determinants or epitopes thatare essentially the same as the antigenic determinant(s) or epitope(s)to which the single variable domains of the invention bind in members ofthe Notch signalling pathway (e.g. in wild-type members of the Notchsignalling pathway). Again, in such a case, the single variable domainsof the invention may bind to such analogs, variants, mutants, alleles,parts and fragments with an affinity and/or specificity that are thesame as, or that are different from (i.e. higher than or lower than),the affinity and specificity with which the single variable domains ofthe invention bind to (wild-type) members of the Notch signallingpathway. It is also included within the scope of the invention that thesingle variable domains of the invention bind to some analogs, variants,mutants, alleles, parts and fragments of the members of the Notchsignalling pathway, but not to others.

When members of the Notch signalling pathway exists in a monomeric formand in one or more multimeric forms, it is within the scope of theinvention that the single variable domains of the invention only bind tomembers of the Notch signalling pathway in monomeric form, only bind tomembers of the Notch signalling pathway in multimeric form, or bind toboth the monomeric and the multimeric form. Again, in such a case, thesingle variable domains of the invention may bind to the monomeric formwith an affinity and/or specificity that are the same as, or that aredifferent from (i.e. higher than or lower than), the affinity andspecificity with which the single variable domains of the invention bindto the multimeric form.

Also, when members of the Notch signalling pathway can associate withother proteins or single variable domains to form protein complexes(e.g. with multiple subunits), it is within the scope of the inventionthat the single variable domains of the invention bind to members of theNotch signalling pathway in its non-associated state, bind to members ofthe Notch signalling pathway in its associated state, or bind to both.In all these cases, the single variable domains of the invention maybind to such multimers or associated protein complexes with an affinityand/or specificity that may be the same as or different from (i.e.higher than or lower than) the affinity and/or specificity with whichthe single variable domains of the invention bind to members of theNotch signalling pathway in its monomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or singlevariable domains that contain two or more single variable domainsdirected against members of the Notch signalling pathway may bind withhigher avidity to members of the Notch signalling pathway than thecorresponding monomeric amino acid sequence(s). For example, and withoutlimitation, proteins or single variable domains that contain two or moresingle variable domains directed against different epitopes of membersof the Notch signalling pathway may (and usually will) bind with higheravidity than each of the different monomers, and proteins or singlevariable domains that contain two or more single variable domainsdirected against members of the Notch signalling pathway may (andusually will) bind also with higher avidity.

Generally, the single variable domains of the invention will at leastbind to those forms of members of the Notch signalling pathway(including monomeric, multimeric and associated forms) that are the mostrelevant from a biological and/or therapeutic point of view, as will beclear to the skilled person.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the singlevariable domains of the invention, and/or to use proteins or singlevariable domains comprising or essentially consisting of one or more ofsuch parts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against members of the Notch signallingpathway; and more preferably will be capable of specific binding tomembers of the Notch signalling pathway, and even more preferablycapable of binding to members of the Notch signalling pathway with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Some non-limiting examples of suchparts, fragments, analogs, mutants, variants, alleles, derivatives,proteins and/or single variable domains will become clear from thefurther description herein. Additional fragments or single variabledomains of the invention may also be provided by suitably combining(i.e. by linking or genetic fusion) one or more (smaller) parts orfragments as described herein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to members of the Notch signalling pathway; and more preferablycapable of binding to members of the Notch signalling pathway with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Also, parts, fragments, analogs,mutants, variants, alleles and/or derivatives of such single variabledomains are preferably such that they comprise an immunoglobulin fold orare capable for forming, under suitable conditions, an immunoglobulinfold.

In particular, but without limitation, the single variable domains ofthe invention may be single variable domains that essentially consist of4 framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The single variable domains of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the single variable domains ofthe invention may be naturally occurring single variable domains (fromany suitable species) or synthetic or semi-synthetic amino acidsequences. In a specific but non-limiting aspect of the invention, theamino acid sequence is a naturally occurring immunoglobulin sequence(from any suitable species) or a synthetic or semi-syntheticimmunoglobulin sequence, including but not limited to “humanized” (asdefined herein) immunoglobulin sequences (such as partially or fullyhumanized mouse or rabbit immunoglobulin sequences, and in particularpartially or fully humanized VHH sequences or Nanobodies), “camelized”(as defined herein) immunoglobulin sequences, as well as immunoglobulinsequences that have been obtained by techniques such as affinitymaturation (for example, starting from synthetic, random or naturallyoccurring immunoglobulin sequences), CDR grafting, veneering, combiningfragments derived from different immunoglobulin sequences, PCR assemblyusing overlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody™ (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence or single variable domain(s) ofthe invention may be a Nanobody® (as defined herein) or a suitablefragment thereof [Note: Nanobody®, Nanobodies® and Nanoclone® areregistered trademarks of Ablynx N.V.]. Such Nanobodies directed againstmembers of the Notch signalling pathway will also be referred to hereinas “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described Nanobodies of the so-called “V_(H)3 class”(i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against members of the Notchsignalling pathway, and for example also covers the Nanobodies belongingto the so-called “V_(H)4 class” (i.e. Nanobodies with a high degree ofsequence homology to human germline sequences of the V_(H)4 class suchas DP-78), as for example described in the U.S. provisional application60/792,279 by Ablynx N.V. entitled “DP-78-like Nanobodies” filed on Apr.14, 2006 (see also PCT/EP2007/003259).

Generally, Nanobodies (in particular VHH sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as further        defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.

More in particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below;    and in which:

-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the single variable domains of SEQ ID NO's: 1    to 22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against members of the Notchsignalling pathway, to suitable fragments thereof, as well as to singlevariable domains that comprise or essentially consist of one or more ofsuch Nanobodies and/or suitable fragments.

SEQ ID NO's [255-269] give the single variable domains of a number ofVHH sequences that have been raised against members of the Notchsignalling pathway.

In particular, the invention in some specific aspects provides:

-   -   single variable domains that are directed against (as defined        herein) members of the Notch signalling pathway and that have at        least 80%, preferably at least 85%, such as 90% or 95% or more        sequence identity with at least one of the single variable        domains of SEQ ID NO's: [255-269] (see Table A-1A for reference        to sequences, same reference to the SEQ ID NO's: [255-269]        applies throughout of the application even if Table A-1A        reference is not given). These single variable domains may        further be such that they neutralize binding of the cognate        ligand to members of the Notch signalling pathway; and/or        compete with the cognate ligand for binding to members of the        Notch signalling pathway; and/or are directed against an        interaction site (as defined herein) on members of the Notch        signalling pathway (such as the ligand binding site);    -   single variable domains that cross-block (as defined herein) the        binding of at least one of the single variable domains of SEQ ID        NO's: [255-269] to members of the Notch signalling pathway        and/or that compete with at least one of the single variable        domains of SEQ ID NO's: [255-269] for binding to members of the        Notch signalling pathway. Again, these single variable domains        may further be such that they neutralize binding of the cognate        ligand to members of the Notch signalling pathway; and/or        compete with the cognate ligand for binding to members of the        Notch signalling pathway; and/or are directed against an        interaction site (as defined herein) on members of the Notch        signalling pathway (such as the ligand binding site);        which single variable domains may be as further described herein        (and may for example be Nanobodies); as well as single variable        domains of the invention that comprise one or more of such        single variable domains (which may be as further described        herein, and may for example be bispecific and/or biparatopic        single variable domains as described herein), and nucleic acid        sequences that encode such single variable domains. Such single        variable domains do not include any naturally occurring ligands.

In some other specific aspects, the invention provides:

-   -   single variable domains of the invention that are specific for        (as defined herein) FcgR3, FcgR1, FcgR2 and pIgR;        which single variable domains of the invention may be as further        described herein (and may for example be Nanobodies); as well as        single variable domains of the invention that comprise one or        more of such single variable domains (which may be as further        described herein, and may for example be bispecific and/or        biparatopic single variable domains as described herein), and        nucleic acid sequences that encode such single variable domains.        Such single variable domains do not include any naturally        occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to members of the Notch signalling pathway and which:

-   i) have at least 80% amino acid identity with at least one of the    single variable domains of SEQ ID NO's: [255-269], in which for the    purposes of determining the degree of amino acid identity, the amino    acid residues that form the CDR sequences are disregarded. In this    respect, reference is also made to Table A-1B (same reference to the    SEQ ID NO's of the frameworks applies throughout of the application    even if Table A-1B reference is not given), which lists the    framework 1 sequences (SEQ ID NO's: [126-143]), framework 2    sequences (SEQ ID NO's: [162-179]), framework 3 sequences (SEQ ID    NO's: [198-215]) and framework 4 sequences (SEQ ID NO's: [234-251])    of the Nanobodies of SEQ ID NO's: [255-269] (with respect to the    amino acid residues at positions 1 to 4 and 27 to 30 of the    framework 1 sequences, reference is also made to the comments made    below. Thus, for determining the degree of amino acid identity,    these residues are preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring VHHsequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be single variable domains thatare as generally defined for Nanobodies in the previous paragraphs, butin which at least one amino acid residue is present (and in particular,in at least one of the framework residues) that is and/or thatcorresponds to a humanizing substitution (as defined herein). Somepreferred, but non-limiting humanizing substitutions (and suitablecombinations thereof) will become clear to the skilled person based onthe disclosure herein. In addition, or alternatively, other potentiallyuseful humanizing substitutions can be ascertained by comparing thesequence of the framework regions of a naturally occurring V_(HH)sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized VHH sequences can be tested for affinity for the target, forstability, for ease and level of expression, and/or for other desiredproperties. In this way, by means of a limited degree of trial anderror, other suitable humanizing substitutions (or suitable combinationsthereof) can be determined by the skilled person based on the disclosureherein. Also, based on the foregoing, (the framework regions of) aNanobody may be partially humanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention arehumanized variants of the Nanobodies of SEQ ID NO's: [255-269].

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can bind (as further defined herein) to members of the Notchsignalling pathway and which:

-   i) are a humanized variant of one of the single variable domains of    SEQ ID NO's: [255-269]; and/or-   ii) have at least 80% amino acid identity with at least one of the    single variable domains of SEQ ID NO's: [255-269] in which for the    purposes of determining the degree of amino acid identity, the amino    acid residues that form the CDR sequences are disregarded;    and in which:-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to members for theNotch signalling pathway. These stretches of amino acid residues may bepresent in, and/or may be corporated into, an amino acid sequence of theinvention, in particular in such a way that they form (part of) theantigen binding site of an amino acid sequence of the invention. Asthese stretches of amino acid residues were first generated as CDRsequences of heavy chain antibodies or VHH sequences that were raisedagainst members of the Notch signalling pathway (or may be based onand/or derived from such CDR sequences, as further described herein),they will also generally be referred to herein as “CDR sequences” (i.e.as CDR1, CDR2 and CDR3, respectively). It should however be noted thatthe invention in its broadest sense is not limited to a specificstructural role or function that these stretches of amino acid residuesmay have in an amino acid sequence of the invention, as long as thesestretches of amino acid residues allow the amino acid sequence of theinvention to bind to members for the Notch signalling pathway. Thus,generally, the invention in its broadest sense comprises any amino acidsequence that is capable of binding to members of the Notch signallingpathway and that comprises one or more CDR sequences as describedherein, and in particular a suitable combination of two or more such CDRsequences, that are suitably linked to each other via one or morefurther amino acid sequences, such that the entire amino acid sequenceforms a binding domain and/or binding unit that is capable of binding tomembers for the Notch signalling pathway. It should however also benoted that the presence of only one such CDR sequence in an amino acidsequence of the invention may by itself already be sufficient to providean amino acid sequence of the invention that is capable of binding tomembers for the Notch signalling pathway, reference is for example againmade to the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1, CDR2 and CDR3 that are described herein in TableA-1B (same reference to the SEQ ID NO's of the CDRs applies throughoutof the application even if Table A-1B reference is not given). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1, CDR2 and CDR3 that aredescribed herein (or any suitable combination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting single variable domains will be clear to the skilledperson, and for example comprise, without limitation, to bindingscaffolds based on or derived from immunoglobulins (i.e. other than theimmunoglobulin sequences already described herein), protein scaffoldsderived from protein A domains (such as Affibodies™), tendamistat,fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrinrepeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol23:1257), and binding moieties based on DNA or RNA including but notlimited to DNA or RNA aptamers (Ulrich et al., Comb Chem High ThroughputScreen 2006 9(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to members for the Notch signalling pathway,and more in particular such that it can bind to members of the Notchsignalling pathway with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein), that is as defined herein.

More in particular, the single variable domains according to this aspectof the invention may be any amino acid sequence that comprises at leastone antigen binding site, wherein said antigen binding site comprises atleast two single variable domains that are chosen from the groupconsisting of the CDR1 described herein, the CDR2 described herein andthe CDR3 described herein, such that (i) when the first amino acidsequence is chosen from the CDR1 described herein, the second amino acidsequence is chosen from the CDR2 described herein or the CDR3 describedherein; (ii) when the first amino acid sequence is chosen from the CDR2described herein, the second amino acid sequence is chosen from the CDR1described herein or the CDR3 described herein; or (iii) when the firstamino acid sequence is chosen from the CDR2 described herein, the secondamino acid sequence is chosen from the CDR1 described herein or the CDR3described herein.

Even more in particular, the single variable domains of the inventionmay be single variable domains that comprise at least one antigenbinding site, wherein said antigen binding site comprises at least threesingle variable domains that are chosen from the group consisting of theCDR1 described herein, the CDR2 described herein and the CDR3 describedherein, such that the first amino acid sequence is chosen from the CDR1described herein, the second amino acid sequence is chosen from the CDR2described herein, and the third amino acid sequence is chosen from theCDR3 described herein. Preferred combinations of CDR1, CDR2 and CDR3will become clear from the further description herein. As will be clearto the skilled person, such an amino acid sequence is preferably animmunoglobulin sequence (as further described herein), but it may forexample also be any other amino acid sequence that comprises a suitablescaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against members for the Notch signallingpathway, that comprises one or more stretches of amino acid residueschosen from the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233];    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more singlevariable domains according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore single variable domains according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more single variable domains according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any single variable domains of the invention thatcomprise one or more single variable domains according to b), c), e),f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the single variable domains of SEQ ID NO's: [144-161];-   ii) the single variable domains of SEQ ID NO's: [180-197]; and-   iii) the single variable domains of SEQ ID NO's: [216-233];    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against members for the Notch signalling pathway.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against members for the Notchsignalling pathway that comprise two or more stretches of amino acidresidues chosen from the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233];    such that (i) when the first stretch of amino acid residues    corresponds to one of the single variable domains according to    a), b) or c), the second stretch of amino acid residues corresponds    to one of the single variable domains according to d), e), f),    g), h) or i); (ii) when the first stretch of amino acid residues    corresponds to one of the single variable domains according to    d), e) or f), the second stretch of amino acid residues corresponds    to one of the single variable domains according to a), b), c),    g), h) or i); or (iii) when the first stretch of amino acid residues    corresponds to one of the single variable domains according to    g), h) or i), the second stretch of amino acid residues corresponds    to one of the single variable domains according to a), b), c),    d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the single variable domains of SEQ ID NO's: [144-161];-   ii) the single variable domains of SEQ ID NO's: [180-197]; and-   iii) the single variable domains of SEQ ID NO's: [216-233];    such that, (i) when the first stretch of amino acid residues    corresponds to one of the single variable domains of SEQ ID NO's:    [144-161], the second stretch of amino acid residues corresponds to    one of the single variable domains of SEQ ID NO's: [180-197] or of    SEQ ID NO's: [216-233]; (ii) when the first stretch of amino acid    residues corresponds to one of the single variable domains of SEQ ID    NO's: [180-197], the second stretch of amino acid residues    corresponds to one of the single variable domains of SEQ ID NO's:    [144-161] or of SEQ ID NO's: [216-233]; or (iii) when the first    stretch of amino acid residues corresponds to one of the single    variable domains of SEQ ID NO's: [216-233], the second stretch of    amino acid residues corresponds to one of the single variable    domains of SEQ ID NO's: [144-161] or of SEQ ID NO's: [180-197].

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against members for the Notch signalling pathway.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against members for the Notchsignalling pathway, that comprises three or more stretches of amino acidresidues, in which the first stretch of amino acid residues is chosenfrom the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233].

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the single variabledomains of SEQ ID NO's: [144-161]; the second stretch of amino acidresidues is chosen from the group consisting of the single variabledomains of SEQ ID NO's: [180-197]; and the third stretch of amino acidresidues is chosen from the group consisting of the single variabledomains of SEQ ID NO's: [216-233].

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against members for the Notch signalling pathway.

Preferred combinations of such stretches of single variable domains willbecome clear from the further disclosure herein.

Preferably, in such single variable domains the CDR sequences have atleast 70% amino acid identity, preferably at least 80% amino acididentity, more preferably at least 90% amino acid identity, such as 95%amino acid identity or more or even essentially 100% amino acid identitywith the CDR sequences of at least one of the single variable domains ofSEQ ID NO's: [255-269]. This degree of amino acid identity can forexample be determined by determining the degree of amino acid identity(in a manner described herein) between said amino acid sequence and oneor more of the sequences of SEQ ID NO's: [255-269], in which the aminoacid residues that form the framework regions are disregarded. Also,such single variable domains of the invention can be as furtherdescribed herein.

Also, such single variable domains are preferably such that they canspecifically bind (as defined herein) to members for the Notchsignalling pathway; and more in particular bind to members of the Notchsignalling pathway with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A) value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];    and/or

CDR2 is chosen from the group consisting of:

-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];    and/or

CDR3 is chosen from the group consisting of:

-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233].

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the single variabledomains of SEQ ID NO's: [144-161]; and/or CDR2 is chosen from the groupconsisting of the single variable domains of SEQ ID NO's: [180-197];and/or CDR3 is chosen from the group consisting of the single variabledomains of SEQ ID NO's: [216-233].

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];    and

CDR2 is chosen from the group consisting of:

-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];    and

CDR3 is chosen from the group consisting of:

-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233]; or any suitable fragment of such an amino acid    sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the single variabledomains of SEQ ID NO's: [144-161]; and CDR2 is chosen from the groupconsisting of the single variable domains of SEQ ID NO's: [180-197]; andCDR3 is chosen from the group consisting of the single variable domainsof SEQ ID NO's: [216-233].

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such single variable domains are preferably such that they canspecifically bind (as defined herein) to members for the Notchsignalling pathway; and more in particular bind to members of the Notchsignalling pathway with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of thesingle variable domains of SEQ ID NO's: [255-269]. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's:[255-269], in which the amino acid residues that form the frameworkregions are disregarded. Such single variable domains of the inventioncan be as further described herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody™ (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the single variabledomains of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody™. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the single variable domains ofthe invention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). For a further description ofthese “Expedite fragments”, reference is again made to WO 03/050531, aswell as to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx N.V.(inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; andHoogenboom, Hendricus Renerus Jacobus Mattheus) filed on Dec. 5, 2006(see also PCT/EP2007/063348).

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moresingle variable domains of the invention (or suitable fragmentsthereof), and optionally further comprises one or more other groups,residues, moieties or binding units. As will become clear to the skilledperson from the further disclosure herein, such further groups,residues, moieties, binding units or single variable domains may or maynot provide further functionality to the amino acid sequence of theinvention (and/or to the compound or construct in which it is present)and may or may not modify the properties of the amino acid sequence ofthe invention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,single variable domains that are suitable for use as a domain antibody,single domain antibodies, single variable domains that are suitable foruse as a single domain antibody, “dAb”'s, single variable domains thatare suitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moresingle variable domains of the invention so as to provide a “derivative”of an amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more singlevariable domains of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

As will be clear from the further description above and herein, thismeans that the single variable domains of the invention can be used as“building blocks” to form single variable domains of the invention, i.e.by suitably combining them with other groups, residues, moieties orbinding units, in order to form compounds or constructs as describedherein (such as, without limitations, the biparatopic. bi/multivalentand bi/multispecific single variable domains of the invention describedherein) which combine within one molecule one or more desired propertiesor biological functions.

The compounds or single variable domains of the invention can generallybe prepared by a method which comprises at least one step of suitablylinking the one or more single variable domains of the invention to theone or more further groups, residues, moieties or binding units,optionally via the one or more suitable linkers, so as to provide thecompound or polypeptide of the invention. Single variable domains of theinvention can also be prepared by a method which generally comprises atleast the steps of providing a nucleic acid that encodes a polypeptideof the invention, expressing said nucleic acid in a suitable manner, andrecovering the expressed polypeptide of the invention. Such methods canbe performed in a manner known per se, which will be clear to theskilled person, for example on the basis of the methods and techniquesfurther described herein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedsingle variable domains form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and singlevariable domains will become clear to the skilled person based on thefurther disclosure herein, and for example comprise single variabledomains or single variable domains of the invention that have beenchemically modified to increase the half-life thereof (for example, bymeans of pegylation); single variable domains of the invention thatcomprise at least one additional binding site for binding to a serumprotein (such as serum albumin); or single variable domains of theinvention that comprise at least one amino acid sequence of theinvention that is linked to at least one moiety (and in particular atleast one amino acid sequence) that increases the half-life of the aminoacid sequence of the invention. Examples of single variable domains ofthe invention that comprise such half-life extending moieties or singlevariable domains will become clear to the skilled person based on thefurther disclosure herein; and for example include, without limitation,single variable domains in which the one or more single variable domainsof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, single variable domains thatare suitable for use as a domain antibody, single domain antibodies,single variable domains that are suitable for use as a single domainantibody, “dAb”'s, single variable domains that are suitable for use asa dAb, or Nanobodies that can bind to serum proteins such as serumalbumin (such as human serum albumin), serum immunoglobulins such asIgG, or transferrin; reference is made to the further description andreferences mentioned herein); single variable domains in which an aminoacid sequence of the invention is linked to an Fc portion (such as ahuman Fc) or a suitable part or fragment thereof; or single variabledomains in which the one or more single variable domains of theinvention are suitable linked to one or more small proteins or peptidesthat can bind to serum proteins (such as, without limitation, theproteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489 and to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx N.V. filed onDec. 5, 2006 (see also PCT/EP2007/063348).

Generally, the compounds or single variable domains of the inventionwith increased half-life preferably have a half-life that is at least1.5 times, preferably at least 2 times, such as at least 5 times, forexample at least 10 times or more than 20 times, greater than thehalf-life of the corresponding amino acid sequence of the invention perse. For example, the compounds or single variable domains of theinvention with increased half-life may have a half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor single variable domains of the invention have a serum half-life thatis increased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or single variable domains of the invention exhibit a serumhalf-life in human of at least about 12 hours, preferably at least 24hours, more preferably at least 48 hours, even more preferably at least72 hours or more. For example, compounds or single variable domains ofthe invention may have a half-life of at least 5 days (such as about 5to 10 days), preferably at least 9 days (such as about 9 to 14 days),more preferably at least about 10 days (such as about 10 to 15 days), orat least about 11 days (such as about 11 to 16 days), more preferably atleast about 12 days (such as about 12 to 18 days or more), or more than14 days (such as about 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such constructs known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or constructs will become clear from the furtherdescription herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or constructs for) modulating members of the Notchsignalling pathway, either in vitro (e.g. in an in vitro or cellularassay) or in vivo (e.g. in an a single cell or in a multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers fromcancer, neurodegenerative diseases and immunomodulatory diseases).

The invention also relates to methods for modulating members of theNotch signalling pathway, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers fromcancer, neurodegenerative diseases and immunomodulatory diseases), whichmethod comprises at least the step of contacting members of the Notchsignalling pathway with at least one amino acid sequence, Nanobody orpolypeptide of the invention, or with a composition comprising the same,in a manner and in an amount suitable to modulate members of the Notchsignalling pathway, with at least one amino acid sequence, Nanobody orpolypeptide of the invention.

The invention also relates to the use of an one amino acid sequence,Nanobody or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulating members ofthe Notch signalling pathway, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers fromcancer, neurodegenerative diseases and immunomodulatory diseases).

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, members of the Notchsignalling pathway, as measured using a suitable in vitro, cellular orin vivo assay (such as those mentioned herein). In particular,“modulating” or “to modulate” may mean either reducing or inhibiting theactivity of, or alternatively increasing the activity of members of theNotch signalling pathway, as measured using a suitable in vitro,cellular or in vivo assay (such as those mentioned herein), by at least1%, preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to activity of members of the Notch signallingpathway in the same assay under the same conditions but without thepresence of the amino acid sequence, Nanobody or polypeptide of theinvention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of members of theNotch signalling pathway for one or more of its targets, ligands orsubstrates; and/or effecting a change (which may either be an increaseor a decrease) in the sensitivity of members of the Notch signallingpathway for one or more conditions in the medium or surroundings inwhich members of the Notch signalling pathway is present (such as pH,ion strength, the presence of co-factors, etc.), compared to the sameconditions but without the presence of the amino acid sequence, Nanobodyor polypeptide of the invention. As will be clear to the skilled person,this may again be determined in any suitable manner and/or using anysuitable assay known per se, such as the assays described herein or inthe prior art cited herein.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which members of the Notch signalling pathway(or in which its substrate(s), ligand(s) or pathway(s) are involved,such as its signalling pathway or metabolic pathway and their associatedbiological or physiological effects) is involved. Again, as will beclear to the skilled person, such an action as an agonist or anantagonist may be determined in any suitable manner and/or using anysuitable (in vitro and usually cellular or in assay) assay known per se,such as the assays described herein or in the prior art cited herein. Inparticular, an action as an agonist or antagonist may be such that anintended biological or physiological activity is increased or decreased,respectively, by at least 1%, preferably at least 5%, such as at least10% or at least 25%, for example by at least 50%, at least 60%, at least70%, at least 80%, or 90% or more, compared to the biological orphysiological activity in the same assay under the same conditions butwithout the presence of the amino acid sequence, Nanobody or polypeptideof the invention.

Modulating may for example involve reducing or inhibiting the binding ofmembers of the Notch signalling pathway to one of its substrates orligands and/or competing with a natural ligand, substrate for binding tomembers of the Notch signalling pathway. Modulating may also involveactivating members of the Notch signalling pathway or the mechanism orpathway in which it is involved. Modulating may be reversible orirreversible, but for pharmaceutical and pharmacological purposes willusually be in a reversible manner.

The invention further relates to methods for preparing or generating theamino acid sequences, single variable domains, nucleic acids, hostcells, products and constructs described herein. Some preferred butnon-limiting examples of such methods will become clear from the furtherdescription herein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of single variable    domains for single variable domains that can bind to and/or have    affinity for members for the Notch signalling pathway;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for members for the Notch signalling pathway.

In such a method, the set, collection or library of single variabledomains may be any suitable set, collection or library of amino acidsequences. For example, the set, collection or library of singlevariable domains may be a set, collection or library of immunoglobulinsequences (as described herein), such as a naïve set, collection orlibrary of immunoglobulin sequences; a synthetic or semi-synthetic set,collection or library of immunoglobulin sequences; and/or a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of singlevariable domains may be a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofsingle variable domains may be a set, collection or library of domainantibodies or single domain antibodies, or may be a set, collection orlibrary of single variable domains that are capable of functioning as adomain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofsingle variable domains may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with members of the Notch signalling pathway orwith a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of single variabledomains may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) single variable domains willbe clear to the person skilled in the art, for example on the basis ofthe further disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating single variable domainscomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for members for the Notch signalling pathway;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withmembers of the Notch signalling pathway or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequencedirected against members of the Notch signalling pathway may comprise atleast the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for members for the    Notch signalling pathway;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding single variable domains may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof single variable domains that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofsingle variable domains may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with members of the Notch signalling pathway or witha suitable antigenic determinant based thereon or derived therefrom,such as an antigenic part, fragment, region, domain, loop or otherepitope thereof. In one particular aspect, said antigenic determinantmay be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of 255-269.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding single variable domains will be clear to the person skilled inthe art, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to single variable domains that are obtainedby the above methods, or alternatively by a method that comprises theone of the above methods and in addition at least the steps ofdetermining the nucleotide sequence or amino acid sequence of saidimmunoglobulin sequence; and of expressing or synthesizing said aminoacid sequence in a manner known per se, such as by expression in asuitable host cell or host organism or by chemical synthesis.

Also, following the steps above, one or more single variable domains ofthe invention may be suitably humanized (or alternatively camelized);and/or the amino acid sequence(s) thus obtained may be linked to eachother or to one or more other suitable single variable domains(optionally via one or more suitable linkers) so as to provide apolypeptide of the invention. Also, a nucleic acid sequence encoding anamino acid sequence of the invention may be suitably humanized (oralternatively camelized) and suitably expressed; and/or one or morenucleic acid sequences encoding an amino acid sequence of the inventionmay be linked to each other or to one or more nucleic acid sequencesthat encode other suitable single variable domains (optionally vianucleotide sequences that encode one or more suitable linkers), afterwhich the nucleotide sequence thus obtained may be suitably expressed soas to provide a polypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, single variable domains, nucleicacids, host cells, products and constructs described herein, as well asto methods for the prevention and/or treatment for diseases anddisorders associated with members for the Notch signalling pathway. Somepreferred but non-limiting applications and uses will become clear fromthe further description herein.

The invention also relates to the amino acid sequences, compounds,constructs, single variable domains, nucleic acids, host cells, productsand compositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences,compounds, constructs, single variable domains, nucleic acids, hostcells, products and compositions described herein for use in therapy ofa disease or disorder that can be prevented or treated by administering,to a subject in need thereof, of (a pharmaceutically effective amountof) an amino acid sequence, compound, construct or polypeptide asdescribed herein.

More in particular, the invention relates to the amino acid sequences,compounds, constructs, single variable domains, nucleic acids, hostcells, products and compositions described herein for use in therapy ofcancer, neurodegenerative diseases and immunomodulatory diseases.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and single variable domains of theinvention comprising the same, which form some of the preferred aspectsof the invention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other single variable domains of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et    al, eds., “Current protocols in molecular biology”, Green Publishing    and Wiley Interscience, New York (1987); Lewin, “Genes II”, John    Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of    Gene Manipulation: An Introduction to Genetic Engineering”, 2nd    edition, University of California Press, Berkeley, Calif. (1981);    Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh    (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.    Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”    (6th Ed.), Garland Science Publishing/Churchill Livingstone, N.Y.    (2005), as well as to the general background art cited herein;-   b) Unless indicated otherwise, the term “immunoglobulin    sequence”—whether used herein to refer to a heavy chain antibody or    to a conventional 4-chain antibody—is used as a general term to    include both the full-size antibody, the individual chains thereof,    as well as all parts, domains or fragments thereof (including but    not limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H)/V_(L) domains, respectively). In addition, the term    “sequence” as used herein (for example in terms like “immunoglobulin    sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)    sequence” or “protein sequence”), should generally be understood to    include both the relevant amino acid sequence as well as nucleic    acids or nucleotide sequences encoding the same, unless the context    requires a more limited interpretation. Also, the term “nucleotide    sequence” as used herein also encompasses a nucleic acid molecule    with said nucleotide sequence, so that the terms “nucleotide    sequence” and “nucleic acid” should be considered equivalent and are    used interchangeably herein;-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residue can generallybe considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).    -   Alternatively, the degree of sequence identity between two or        more nucleotide sequences may be calculated using a known        computer algorithm for sequence alignment such as NCBI Blast        v2.0, using standard settings.    -   Some other techniques, computer algorithms and settings for        determining the degree of sequence identity are for example        described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO        00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two nucleotide sequences in        accordance with the calculation method outlined hereinabove, the        nucleotide sequence with the greatest number of nucleotides will        be taken as the “first” nucleotide sequence, and the other        nucleotide sequence will be taken as the “second” nucleotide        sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.    -   Alternatively, the degree of sequence identity between two        single variable domains may be calculated using a known computer        algorithm, such as those mentioned above for determining the        degree of sequence identity for nucleotide sequences, again        using standard settings.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two single variable domains in        accordance with the calculation method outlined hereinabove, the        amino acid sequence with the greatest number of amino acid        residues will be taken as the “first” amino acid sequence, and        the other amino acid sequence will be taken as the “second”        amino acid sequence.    -   Also, in determining the degree of sequence identity between two        amino acid sequences, the skilled person may take into account        so-called “conservative” amino acid substitutions, which can        generally be described as amino acid substitutions in which an        amino acid residue is replaced with another amino acid residue        of similar chemical structure and which has little or        essentially no influence on the function, activity or other        biological properties of the polypeptide. Such conservative        amino acid substitutions are well known in the art, for example        from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and        WO 01/09300; and (preferred) types and/or combinations of such        substitutions may be selected on the basis of the pertinent        teachings from WO 04/037999 as well as WO 98/49185 and from the        further references cited therein.    -   Such conservative substitutions preferably are substitutions in        which one amino acid within the following groups (a)-(e) is        substituted by another amino acid residue within the same        group: (a) small aliphatic, nonpolar or slightly polar residues:        Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged        residues and their (uncharged) amides: Asp, Asn, Glu and        Gln; (c) polar, positively charged residues: His, Arg and        Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val        and Cys; and (e) aromatic residues: Phe, Tyr and Trp.    -   Particularly preferred conservative substitutions are as        follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or        into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into        Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile        into Leu or into Val; Leu into Ile or into Val; Lys into Arg,        into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe        into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp        into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into        Leu.    -   Any amino acid substitutions applied to the single variable        domains described herein may also be based on the analysis of        the frequencies of amino acid variations between homologous        proteins of different species developed by Schulz et al.,        Principles of Protein Structure, Springer-Verlag, 1978, on the        analyses of structure forming potentials developed by Chou and        Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47:        45-149, 1978, and on the analysis of hydrophobicity patterns in        proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci.        USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157:        105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15:        321-353, 1986, all incorporated herein in their entirety by        reference. Information on the primary, secondary and tertiary        structure of Nanobodies is given in the description herein and        in the general background art cited above. Also, for this        purpose, the crystal structure of a V_(HH) domain from a llama        is for example given by Desmyter et al., Nature Structural        Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural        Structural Biology (1996); 3, 752-757; and Decanniere et al.,        Structure, Vol. 7, 4, 361 (1999). Further information about some        of the amino acid residues that in conventional V_(H) domains        form the V_(H)/V_(L) interface and potential camelizing        substitutions on these positions can be found in the prior art        cited above.-   g) nucleic acid sequences are said to be “exactly the same” if they    have 100% sequence identity (as defined herein) over their entire    length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two single    variable domains can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the firstmentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the    firstmentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the firstmentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when a Nanobody of the invention    is said to comprise a CDR sequence, this may mean that said CDR    sequence has been incorporated into the Nanobody of the invention,    but more usually this generally means that the Nanobody of the    invention contains within its sequence a stretch of amino acid    residues with the same amino acid sequence as said CDR sequence,    irrespective of how said Nanobody of the invention has been    generated or obtained. It should also be noted that when the latter    amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the firstmentioned    amino acid sequence (in other words, the firstmentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a Nanobody of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said Nanobody, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the firstmentioned nucleotide sequence is preferably such that, when    it is expressed into an expression product (e.g. a polypeptide), the    amino acid sequence encoded by the latter nucleotide sequence forms    part of said expression product (in other words, that the latter    nucleotide sequence is in the same reading frame as the    firstmentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino acid sequence is considered to    be “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an amino acid sequence (such as an antibody chain, and in    particular to a globular region of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Usually, such a domain will comprise peptide loops (for example 3 or    4 peptide loops) stabilized, for example, as a sheet or by disulfide    bonds. The term “binding domain” refers to such a domain that is    directed against an antigenic determinant (as defined herein);-   l) The term “antigenic determinant” refers to the epitope on the    antigen recognized by the antigen-binding molecule (such as a    Nanobody or a polypeptide of the invention) and more in particular    by the antigen-binding site of said molecule. The terms “antigenic    determinant” and “epitope” may also be used interchangeably herein.-   m) An amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    Nanobody or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (K_(D)), is a measure for the binding    strength between an antigenic determinant and an antigen-binding    site on the antigen-binding protein: the lesser the value of the    K_(D), the stronger the binding strength between an antigenic    determinant and the antigen-binding molecule (alternatively, the    affinity can also be expressed as the affinity constant (K_(A)),    which is 1/K_(D)). As will be clear to the skilled person (for    example on the basis of the further disclosure herein), affinity can    be determined in a manner known per se, depending on the specific    antigen of interest. Avidity is the measure of the strength of    binding between an antigen-binding molecule (such as a Nanobody or    polypeptide of the invention) and the pertinent antigen. Avidity is    related to both the affinity between an antigenic determinant and    its antigen binding site on the antigen-binding molecule and the    number of pertinent binding sites present on the antigen-binding    molecule. Typically, antigen-binding proteins (such as the amino    acid sequences, Nanobodies and/or single variable domains of the    invention) will bind to their antigen with a dissociation constant    (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to    10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²    moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to    10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or    more and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        then 10⁻⁴ moles/liter or 10⁻³ moles/liter (e.g., of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].    -   The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, Nanobody or polypeptide of the invention and its        intended target) will mainly be expressed in terms of the K_(D)        value of their interaction; it being clear to the skilled person        that in view of the relation K_(A)=1/K_(D), specifying the        strength of molecular interaction by its K_(D) value can also be        used to calculate the corresponding K_(A) value. The K_(D)-value        characterizes the strength of a molecular interaction also in a        thermodynamic sense as it is related to the free energy (DG) of        binding by the well known relation DG=RT·ln(K_(D)) (equivalently        DG=−RT·ln(K_(A))), where R equals the gas constant, T equals the        absolute temperature and ln denotes the natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M        (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹ s⁻¹. The on-rate may vary between 10² M⁻¹ s⁻¹ to        about 10⁷ M⁻¹ s⁻¹, approaching the diffusion-limited association        rate constant for bimolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between        10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1 s⁻¹ (t_(1/2)=0.69 s).    -   The affinity of a molecular interaction between two molecules        can be measured via different techniques known per se, such as        the well known surface plasmon resonance (SPR) biosensor        technique (see for example Ober et al., Intern. Immunology, 13,        1551-1559, 2001) where one molecule is immobilized on the        biosensor chip and the other molecule is passed over the        immobilized molecule under flow conditions yielding k_(on),        k_(off) measurements and hence K_(D) (or K_(A)) values. This can        for example be performed using the well-known BIACORE        instruments.    -   It will also be clear to the skilled person that the measured        K_(D) may correspond to the apparent K_(D) if the measuring        process somehow influences the intrinsic binding affinity of the        implied molecules for example by artefacts related to the        coating on the biosensor of one molecule. Also, an apparent        K_(D) may be measured if one molecule contains more than one        recognition sites for the other molecule. In such situation the        measured affinity may be affected by the avidity of the        interaction by the two molecules.    -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic.    -   However, the accurate measurement of K_(D) may be quite        labor-intensive and as consequence, often apparent K_(D) values        are determined to assess the binding strength of two molecules.        It should be noted that as long all measurements are made in a        consistent way (e.g. keeping the assay conditions unchanged)        apparent K_(D) measurements can be used as an approximation of        the true K_(D) and hence in the present document K_(D) and        apparent K_(D) should be treated with equal importance or        relevance. Finally, it should be noted that in many situations        the experienced scientist may judge it to be convenient to        determine the binding affinity relative to some reference        molecule. For example, to assess the binding strength between        molecules A and B, one may e.g. use a reference molecule C that        is known to bind to B and that is suitably labelled with a        fluorophore or chromophore group or other chemical moiety, such        as biotin for easy detection in an ELISA or FACS (Fluorescent        activated cell sorting) or other format (the fluorophore for        fluorescence detection, the chromophore for light absorption        detection, the biotin for streptavidin-mediated ELISA        detection). Typically, the reference molecule C is kept at a        fixed concentration and the concentration of A is varied for a        given concentration or amount of B. As a result an IC₅₀ value is        obtained corresponding to the concentration of A at which the        signal measured for C in absence of A is halved. Provided        K_(D ref), the K_(D) of the reference molecule, is known, as        well as the total concentration c_(ref) of the reference        molecule, the apparent K_(D) for the interaction A-B can be        obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomolgus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as to the standard handbooks, such as Kenneth, A et    al: Chemical Stability of Pharmaceuticals: A Handbook for    Pharmacists and Peters et al, Pharmacokinete analysis: A Practical    Approach (1996). Reference is also made to “Pharmacokinetics”, M    Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition    (1982).    -   As will also be clear to the skilled person (see for example        pages 6 and 7 of WO 04/003019 and in the further references        cited therein), the half-life can be expressed using parameters        such as the t1/2-alpha, t1/2-beta and the area under the curve        (AUC). In the present specification, an “increase in half-life”        refers to an increase in any one of these parameters, such as        any two of these parameters, or essentially all three these        parameters. As used herein “increase in half-life” or “increased        half-life” in particular refers to an increase in the t1/2-beta,        either with or without an increase in the t1/2-alpha and/or the        AUC or both.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.    -   As will be clear to the skilled person, “modulating” may also        involve effecting a change (which may either be an increase or a        decrease) in affinity, avidity, specificity and/or selectivity        of a target or antigen for one or more of its ligands, binding        partners, partners for association into a homomultimeric or        heteromultimeric form, or substrates; and/or effecting a change        (which may either be an increase or a decrease) in the        sensitivity of the target or antigen for one or more conditions        in the medium or surroundings in which the target or antigen is        present (such as pH, ion strength, the presence of co-factors,        etc.), compared to the same conditions but without the presence        of the construct of the invention. As will be clear to the        skilled person, this may again be determined in any suitable        manner and/or using any suitable assay known per se, depending        on the target or antigen involved.    -   “Modulating” may also mean effecting a change (i.e. an activity        as an agonist, as an antagonist or as a reverse agonist,        respectively, depending on the target or antigen and the desired        biological or physiological effect) with respect to one or more        biological or physiological mechanisms, effects, responses,        functions, pathways or activities in which the target or antigen        (or in which its substrate(s), ligand(s) or pathway(s) are        involved, such as its signalling pathway or metabolic pathway        and their associated biological or physiological effects) is        involved. Again, as will be clear to the skilled person, such an        action as an agonist or an antagonist may be determined in any        suitable manner and/or using any suitable (in vitro and usually        cellular or in assay) assay known per se, depending on the        target or antigen involved. In particular, an action as an        agonist or antagonist may be such that an intended biological or        physiological activity is increased or decreased, respectively,        by at least 1%, preferably at least 5%, such as at least 10% or        at least 25%, for example by at least 50%, at least 60%, at        least 70%, at least 80%, or 90% or more, compared to the        biological or physiological activity in the same assay under the        same conditions but without the presence of the construct of the        invention.    -   Modulating may for example also involve allosteric modulation of        the target or antigen; and/or reducing or inhibiting the binding        of the target or antigen to one of its substrates or ligands        and/or competing with a natural ligand, substrate for binding to        the target or antigen. Modulating may also involve activating        the target or antigen or the mechanism or pathway in which it is        involved. Modulating may for example also involve effecting a        change in respect of the folding or confirmation of the target        or antigen, or in respect of the ability of the target or        antigen to fold, to change its confirmation (for example, upon        binding of a ligand), to associate with other (sub)units, or to        disassociate. Modulating may for example also involve effecting        a change in the ability of the target or antigen to transport        other compounds or to serve as a channel for other compounds        (such as ions).    -   Modulating may be reversible or irreversible, but for        pharmaceutical and pharmacological purposes will usually be in a        reversible manner.-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10,000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10,000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide. Preferably, when an amino acid    sequence or polypeptide is “specific for” a first target or antigen    compared to a second target or antigen, it is directed against (as    defined herein) said first target or antigen, but not directed    against said second target or antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an amino acid    sequence or other binding agents (such as a polypeptide of the    invention) to interfere with the binding of other single variable    domains or binding agents of the invention to a given target, herein    against a member of the Notch signalling pathway. The extend to    which an amino acid sequence or other binding agents of the    invention is able to interfere with the binding of another to a    member of the Notch signalling pathway, and therefore whether it can    be said to cross-block according to the invention, can be determined    using competition binding assays. One particularly suitable    quantitative assay uses a Biacore machine which can measure the    extent of interactions using surface plasmon resonance technology.    Another suitable quantitative cross-blocking assay uses an    ELISA-based approach to measure competition between amino acid    sequence or another binding agent in terms of their binding to the    target.    -   The following generally describes a suitable Biacore assay for        determining whether an amino acid sequence or other binding        agent cross-blocks or is capable of cross-blocking according to        the invention. It will be appreciated that the assay can be used        with any of the amino acid sequence or other binding agents        described herein. The Biacore machine (for example the        Biacore 3000) is operated in line with the manufacturer's        recommendations. Thus in one cross-blocking assay, the target        protein is coupled to a CM5 Biacore chip using standard amine        coupling chemistry to generate a surface that is coated with the        target. Typically 200-800 resonance units of the target would be        coupled to the chip (an amount that gives easily measurable        levels of binding but that is readily saturable by the        concentrations of test reagent being used). Two test single        variable domains (termed A* and B*) to be assessed for their        ability to cross-block each other are mixed at a one to one        molar ratio of binding sites in a suitable buffer to create the        test mixture. When calculating the concentrations on a binding        site basis the molecular weight of an amino acid sequence is        assumed to be the total molecular weight of the amino acid        sequence divided by the number of target binding sites on that        amino acid sequence. The concentration of each amino acid        sequence in the test mix should be high enough to readily        saturate the binding sites for that amino acid sequence on the        target molecules captured on the Biacore chip. The single        variable domains in the mixture are at the same molar        concentration (on a binding basis) and that concentration would        typically be between 1.00 and 1.5 micro-molar (on a binding site        basis). Separate solutions containing A* alone and B* alone are        also prepared. A* and B* in these solutions should be in the        same buffer and at the same concentration as in the test mix.        The test mixture is passed over the target-coated Biacore chip        and the total amount of binding recorded. The chip is then        treated in such a way as to remove the bound single variable        domains without damaging the chip-bound target. Typically this        is done by treating the chip with 30 mM HCl for 60 seconds. The        solution of A* alone is then passed over the target-coated        surface and the amount of binding recorded. The chip is again        treated to remove all of the bound single variable domains        without damaging the chip-bound target. The solution of B* alone        is then passed over the target-coated surface and the amount of        binding recorded. The maximum theoretical binding of the mixture        of A* and B* is next calculated, and is the sum of the binding        of each amino acid sequence when passed over the target surface        alone. If the actual recorded binding of the mixture is less        than this theoretical maximum then the two single variable        domains are cross-blocking each other. Thus, in general, a        cross-blocking amino acid sequence or other binding agent        according to the invention is one which will bind to the target        in the above Biacore cross-blocking assay such that during the        assay and in the presence of a second amino acid sequence or        other binding agent of the invention the recorded binding is        between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical        binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of        the maximum theoretical binding, and more specifically between        70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as        just defined above) of the two single variable domains or        binding agents in combination. The Biacore assay described above        is a primary assay used to determine if single variable domains        or other binding agents cross-block each other according to the        invention. On rare occasions particular single variable domains        or other binding agents may not bind to target coupled via amine        chemistry to a CM5 Biacore chip (this usually occurs when the        relevant binding site on target is masked or destroyed by the        coupling to the chip). In such cases cross-blocking can be        determined using a tagged version of the target, for example a        N-terminal His-tagged version (R & D Systems, Minneapolis,        Minn., USA; 2005 cat#1406-ST-025). In this particular format, an        anti-His amino acid sequence would be coupled to the Biacore        chip and then the His-tagged target would be passed over the        surface of the chip and captured by the anti-His amino acid        sequence. The cross blocking analysis would be carried out        essentially as described above, except that after each chip        regeneration cycle, new His-tagged target would be loaded back        onto the anti-His amino acid sequence coated surface. In        addition to the example given using N-terminal His-tagged        [target], C-terminal His-tagged target could alternatively be        used. Furthermore, various other tags and tag binding protein        combinations that are known in the art could be used for such a        cross-blocking analysis (e.g. HA tag with anti-HA antibodies;        FLAG tag with anti-FLAG antibodies; biotin tag with        streptavidin).    -   The following generally describes an ELISA assay for determining        whether an amino acid sequence or other binding agent directed        against a target cross-blocks or is capable of cross-blocking as        defined herein. It will be appreciated that the assay can be        used with any of the single variable domains (or other binding        agents such as single variable domains of the invention)        described herein. The general principal of the assay is to have        an amino acid sequence or binding agent that is directed against        the target coated onto the wells of an ELISA plate. An excess        amount of a second, potentially cross-blocking, anti-target        amino acid sequence is added in solution (i.e. not bound to the        ELISA plate). A limited amount of the target is then added to        the wells. The coated amino acid sequence and the amino acid        sequence in solution compete for binding of the limited number        of target molecules. The plate is washed to remove excess target        that has not been bound by the coated amino acid sequence and to        also remove the second, solution phase amino acid sequence as        well as any complexes formed between the second, solution phase        amino acid sequence and target. The amount of bound target is        then measured using a reagent that is appropriate to detect the        target. An amino acid sequence in solution that is able to        cross-block the coated amino acid sequence will be able to cause        a decrease in the number of target molecules that the coated        amino acid sequence can bind relative to the number of target        molecules that the coated amino acid sequence can bind in the        absence of the second, solution phase, amino acid sequence. In        the instance where the first amino acid sequence, e.g. an Ab-X,        is chosen to be the immobilized amino acid sequence, it is        coated onto the wells of the ELISA plate, after which the plates        are blocked with a suitable blocking solution to minimize        non-specific binding of reagents that are subsequently added. An        excess amount of the second amino acid sequence, i.e. Ab-Y, is        then added to the ELISA plate such that the moles of Ab-Y target        binding sites per well are at least 10 fold higher than the        moles of Ab-X target binding sites that were used, per well,        during the coating of the ELISA plate. Target is then added such        that the moles of target added per well are at least 25-fold        lower than the moles of Ab-X target binding sites that were used        for coating each well. Following a suitable incubation period        the ELISA plate is washed and a reagent for detecting the target        is added to measure the amount of target specifically bound by        the coated anti-target amino acid sequence (in this case Ab-X).        The background signal for the assay is defined as the signal        obtained in wells with the coated amino acid sequence (in this        case Ab-X), second solution phase amino acid sequence (in this        case Ab-Y), target buffer only (i.e. no target) and target        detection reagents. The positive control signal for the assay is        defined as the signal obtained in wells with the coated amino        acid sequence (in this case Ab-X), second solution phase amino        acid sequence buffer only (i.e. no second solution phase amino        acid sequence), target and target detection reagents. The ELISA        assay may be run in such a manner so as to have the positive        control signal be at least 6 times the background signal. To        avoid any artefacts (e.g. significantly different affinities        between Ab-X and Ab-Y for [target]) resulting from the choice of        which amino acid sequence to use as the coating amino acid        sequence and which to use as the second (competitor) amino acid        sequence, the cross-blocking assay may to be run in two        formats: 1) format 1 is where Ab-X is the amino acid sequence        that is coated onto the ELISA plate and Ab-Y is the competitor        amino acid sequence that is in solution and 2) format 2 is where        Ab-Y is the amino acid sequence that is coated onto the ELISA        plate and Ab-X is the competitor amino acid sequence that is in        solution. Ab-X and Ab-Y are defined as cross-blocking if, either        in format 1 or in format 2, the solution phase anti-target amino        acid sequence is able to cause a reduction of between 60% and        100%, specifically between 70% and 100%, and more specifically        between 80% and 100%, of the target detection signal {i.e. the        amount of target bound by the coated amino acid sequence) as        compared to the target detection signal obtained in the absence        of the solution phase anti-target amino acid sequence (i.e. the        positive control wells).-   t) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   u) The amino acid residues of a Nanobody are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication); or referred to herein.    According to this numbering, FR1 of a Nanobody comprises the amino    acid residues at positions 1-30, CDR1 of a Nanobody comprises the    amino acid residues at positions 31-35, FR2 of a Nanobody comprises    the amino acids at positions 36-49, CDR2 of a Nanobody comprises the    amino acid residues at positions 50-65, FR3 of a Nanobody comprises    the amino acid residues at positions 66-94, CDR3 of a Nanobody    comprises the amino acid residues at positions 95-102, and FR4 of a    Nanobody comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position 1 according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FR4 and vice versa.].    -   Alternative methods for numbering the amino acid residues of        V_(H) domains, which methods can also be applied in an analogous        manner to V_(HH) domains from Camelids and to Nanobodies, are        the method described by Chothia et al. (Nature 342, 877-883        (1989)), the so-called “AbM definition” and the so-called        “contact definition”. However, in the present description,        claims and figures, the numbering according to Kabat as applied        to V_(HH) domains by Riechmann and Muyldermans will be followed,        unless indicated otherwise; and-   v) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74 (2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spatial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and Nanobodies can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and Nanobodies can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and Nanobodies are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived “dAb's”        described by Ward et al., Nature, Vol. 341, 1989, p. 544);    -   V_(HH) domains and Nanobodies are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and Nanobodies are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, Nanobodies and proteins/single variable domains        containing the same can be produced using microbial fermentation        (e.g. as further described below) and do not require the use of        mammalian expression systems, as with for example conventional        antibody fragments;    -   V_(HH) domains and Nanobodies are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) than such conventional 4-chain        antibodies and antigen binding fragments thereof;    -   V_(HH) domains and Nanobodies can show so-called cavity-binding        properties (inter alia due to their extended CDR3 loop, compared        to conventional V_(H) domains) and can therefore also access        targets and epitopes not accessible to conventional 4-chain        antibodies and antigen-binding fragments thereof. For example,        it has been shown that V_(HH) domains and Nanobodies can inhibit        enzymes (see for example WO 97/49805; Transue et al., Proteins        1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998 Jul.        1; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides Nanobodiesagainst members for the Notch signalling pathway, and in particularNanobodies against members of the Notch signalling pathway from awarm-blooded animal, and more in particular Nanobodies against membersof the Notch signalling pathway from a mammal, and especially Nanobodiesagainst human members for the Notch signalling pathway, as well asproteins and/or single variable domains comprising at least one suchNanobody.

In particular, the invention provides Nanobodies against members for theNotch signalling pathway, and proteins and/or single variable domainscomprising the same, that have improved therapeutic and/orpharmacological properties and/or other advantageous properties (suchas, for example, improved ease of preparation and/or reduced costs ofgoods), compared to conventional antibodies against members of the Notchsignalling pathway or fragments thereof, compared to constructs thatcould be based on such conventional antibodies or antibody fragments(such as Fab′ fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies”and other multispecific constructs (see for example the review byHolliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36)),and also compared to the so-called “dAb's” or similar (single) domainantibodies that may be derived from variable domains of conventionalantibodies. These improved and advantageous properties will become clearfrom the further description herein, and for example include, withoutlimitation, one or more of:

-   -   increased affinity and/or avidity for members for the Notch        signalling pathway, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased specificity towards members for the Notch signalling        pathway, either in a monovalent format, in a multivalent format        (for example in a bivalent format) and/or in a multispecific        format (for example one of the multispecific formats described        hereinbelow);    -   decreased or where desired increased cross-reactivity with        members of the Notch signalling pathway from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow).

As generally described herein for the single variable domains of theinvention, the Nanobodies of the invention are preferably in essentiallyisolated form (as defined herein), or form part of a protein orpolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more Nanobodies of the invention and whichmay optionally further comprise one or more further single variabledomains (all optionally linked via one or more suitable linkers). Forexample, and without limitation, the one or more single variable domainsof the invention may be used as a binding unit in such a protein orpolypeptide, which may optionally contain one or more further singlevariable domains that can serve as a binding unit (i.e. against one ormore other targets than members for the Notch signalling pathway), so asto provide a monovalent, multivalent or multispecific polypeptide of theinvention, respectively, all as described herein. In particular, such aprotein or polypeptide may comprise or essentially consist of one ormore Nanobodies of the invention and optionally one or more (other)Nanobodies (i.e. directed against other targets than members for theNotch signalling pathway), all optionally linked via one or moresuitable linkers, so as to provide a monovalent, multivalent ormultispecific Nanobody construct, respectively, as further describedherein. Such proteins or single variable domains may also be inessentially isolated form (as defined herein).

In a Nanobody of the invention, the binding site for binding againstmembers of the Notch signalling pathway is preferably formed by the CDRsequences. Optionally, a Nanobody of the invention may also, and inaddition to the at least one binding site for binding against membersfor the Notch signalling pathway, contain one or more further bindingsites for binding against other antigens, proteins or targets. Formethods and positions for introducing such second binding sites,reference is for example made to Keck and Huston, Biophysical Journal,71, October 1996, 2002-2011; EP 0 640 130; and WO 06/07260.

As generally described herein for the single variable domains of theinvention, when a Nanobody of the invention (or a polypeptide of theinvention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against human members forthe Notch signalling pathway; whereas for veterinary purposes, it ispreferably directed against members of the Notch signalling pathway fromthe species to be treated. Also, as with the single variable domains ofthe invention, a Nanobody of the invention may or may not becross-reactive (i.e. directed against members of the Notch signallingpathway from two or more species of mammal, such as against humanmembers of the Notch signalling pathway and members of the Notchsignalling pathway from at least one of the species of mammal mentionedherein).

Also, again as generally described herein for the single variabledomains of the invention, the Nanobodies of the invention may generallybe directed against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of members for the Notchsignalling pathway. However, it is generally assumed and preferred thatthe Nanobodies of the invention (and single variable domains comprisingthe same) are directed against the receptor-ligand binding epitopes ofthe members of the Notch signalling pathway.

As already described herein, the amino acid sequence and structure of aNanobody can be considered—without however being limited thereto—to becomprised of four framework regions or “FR's” (or sometimes alsoreferred to as “FW's”), which are referred to in the art and herein as“Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the Nanobodies of the invention are as described herein.Other suitable CDR sequences can be obtained by the methods describedherein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to members of the Notch signalling        pathway with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²        moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or        less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an        association constant (K_(A)) of 10⁵ to 10¹² liter/moles or more,        and preferably 10⁷ to 10¹² liter/moles or more and more        preferably 10⁸ to 10¹² liter/moles);        and/or such that:    -   the Nanobodies can bind to members of the Notch signalling        pathway with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about 10⁷        M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more        preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between        10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   the Nanobodies can bind to members of the Notch signalling        pathway with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s) and        10⁻⁶ s⁻¹ (providing a near irreversible complex with a t_(1/2)        of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,        more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of theinvention are such that: a monovalent Nanobody of the invention (or apolypeptide that contains only one Nanobody of the invention) ispreferably such that it will bind to members of the Notch signallingpathway with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM.

The affinity of the Nanobody of the invention against members of theNotch signalling pathway can be determined in a manner known per se, forexample using the general techniques for measuring K_(D). K_(A), k_(off)or k_(on) mentioned herein, as well as some of the specific assaysdescribed herein.

Some preferred IC50 values for binding of the Nanobodies of theinvention (and of single variable domains comprising the same) tomembers of the Notch signalling pathway will become clear from thefurther description and examples herein.

In a preferred but non-limiting aspect, the invention relates to aNanobody (as defined herein) against members for the Notch signallingpathway, which consists of 4 framework regions (FR1 to FR4 respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which:

CDR1 is chosen from the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];    and/or

CDR2 is chosen from the group consisting of:

-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];    and/or

CDR3 is chosen from the group consisting of:

-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233];    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) against members forthe Notch signalling pathway, which consists of 4 framework regions (FR1to FR4 respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which:

CDR1 is chosen from the group consisting of:

-   a) the single variable domains of SEQ ID NO's: [144-161];-   b) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [144-161];-   c) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [144-161];    and

CDR2 is chosen from the group consisting of:

-   d) the single variable domains of SEQ ID NO's: [180-197];-   e) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [180-197];-   f) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [180-197];    and

CDR3 is chosen from the group consisting of:

-   g) the single variable domains of SEQ ID NO's: [216-233];-   h) single variable domains that have at least 80% amino acid    identity with at least one of the single variable domains of SEQ ID    NO's: [216-233];-   i) single variable domains that have 3, 2, or 1 amino acid    difference with at least one of the single variable domains of SEQ    ID NO's: [216-233];    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the single variable domains of theinvention, when a Nanobody of the invention contains one or more 144-161according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when a Nanobody of the invention contains one or more 180-197according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or more216-233 according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any Nanobody of the invention that comprises one or more 144-161,180-197 and/or 216-233 according to b), c), e), f), h) or i),respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table A-1B below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) Nanobodies of the invention. As will beclear to the skilled person, a combination of CDR1, CDR2 and CDR3 thatoccur in the same clone (i.e. CDR1, CDR2 and CDR3 that are mentioned onthe same line in Table A-1B) will usually be preferred (although theinvention in its broadest sense is not limited thereto, and alsocomprises other suitable combinations of the CDR sequences mentioned inTable A-1B). Also, a combination of CDR sequences and frameworksequences that occur in the same clone (i.e. CDR sequences and frameworksequences that are mentioned on the same line in Table A-1B) willusually be preferred (although the invention in its broadest sense isnot limited thereto, and also comprises other suitable combinations ofthe CDR sequences and framework sequences mentioned in Table A-1B, aswell as combinations of such CDR sequences and other suitable frameworksequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinationsof CDR's mentioned in Table A-1B, each CDR can be replaced by a CDRchosen from the group consisting of single variable domains that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withthe mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table A-1B,    a conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table A-1B;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table    A-1B.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table A-1B will generally be preferred.

TABLE A-1A Preferred single variable domains (Nanobodies ™). Clone SEQID name Target NO: Preferred single variable domains(Nanobodies ™). >179E6 DLL4 329EVQLVESGGGLVQAGGSLRLSCIASGDTSEIYDMGWFRQAPGKEREFVSSIHWRGRGTNYTDSVEGRFTISKDNAKNMVYLQMNSLKPEDTAVYYCAATRLIVYTPTGFRQYFDWGQGAQVTVSS >179H9 DLL4 330EVQLVESGGGLVQAGGSLRLSCAASG??F?SDVMGWFRRAPGKEREFVASITTTGNEYYEDSLKGRFTVSRDIAENTMYLEMTNLKPEDTAEYSCAGRLLGSTIRSHEYRYWGQGTQVTVSS >179A10 DLL4 331EVQLVESGGGLVQAGGSLRLSCVASGDTSEIYDMGWFRQAPGKEREFVSSIHWGGRGTNYTDSVKGRFTISKDNAKNMVHLQMNSLKPEDTAVYYCAATRLIVYTPTGFRQYFDWGQGTQVTVSS >179D12 DLL4 332EVQLVESGGGLVQAGGSLRLSCVASGDTSEIYDMGWFRQAPGKEREFVSSIHWGGRGTNYTDSVKGRFTISKDNAKNMVYLQMNSLKPEDTAVYYCAATRLIVYTPTGFRQYFDWGQGTQVTVSS >179F6 DLL4 333EVQLVESGGGLVQAGGSLRLSCAASGSDFSGFLLGWYRQPPGKQRELIAQTTDGGRTNYGDSVKGRFTISRDNAKNTWYLQMDSLKPEDTGVYYCNTFPLTSWGQGTQVTVSS >179F7 DLL4 334EVQLVESGGGLVQSGGSLSLSCAASGSVLRINDMGWYRQAPGKTREMVATITRSGVLNYTDSVKGRFTVSRDDAKNTVYLQMSSLKPEDTAVYSCFANIVIRSSGYFNRYWGQGTLVTVSS >178C4 Jagged-1 335EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCMRGRDGSTYYADSVNGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAHTDFACYIGYYSDYDPHDYWGQGTQVTVSS >178A7 Jagged-1 336EVQLVESGGGLVQPGGSLRLSCATSGVIFSTAAMGWYRQAPGKSRVLVARMFSDGRTIYAESVKRRFTISRDNAKNTVYLQMNSLEPEDTAVYYCNALQFGAVYWGQGTQVTVSS >178D9 Jagged-1 337EVQLVKSGGGLVQPGGSLRLSCAASGSIVSANLIGWYRQAPGKQREGVAFITSGGAINYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAIYYCNLRQLGNVYWAQGTQVTVSS >178C11 Jagged-1 338EVQLVESGGGLVQPGGSLRLSCATSGVIFSTAAMGWYRQAPGKSRVLVARMFSDGRTIYAESVKRRFTISRDNAKNTVYLQMNSLEPEDTAVYYCNALQFGAVYWGQGTQVTVSS >180A4 Notch-1 339EVQLVESGGGLVQPGGSLRLSCAASGRTSSPIAMGWFRQAPGKEREFVSGITWSGAYTHYANSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCTTATNSTTGYDYWGQGTQVTVSS >180B1 Notch-1 340EVQLVESGGDLVQPGGSLRLSCAASGTVFSNNDMGWSRQAPGKERELVASFTSGGNTVYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDLLRGKLYWGQGTQVTVSS >180B3 Notch-1 341EVQLVESGGDLVQPGGSLRLSCAASGTVFSNNDMGWSRQAPGKERELVASFTSGGNTVYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDLLRGKLYWGQGTQVTVSS >180C1 Notch-1 342EVQLVESGGDLVQPGGSLRLSCAASRTVFSISDMGWSRQAPGKERELVASISSDNYTVYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS >180C7 Notch-1 343EVQLVESGGGLVQAGGSLRLSCAASGGIFGINAVAWYRQAPGKERDWVALIVGEAITRYTDSVSGRFTISRDNAKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS >180F8 Notch-1 344EVQLVESGGDLVQPGGSLRLSCTTSGTVFSINDMGWSRQAPGKGRELVASISSEGTTIYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS >180F4 Notch-1 345EVQLVESGGGLVQAGDSLRLSCAVSGGSFSSYTMGWVRQAPGKEREAVASIWRSGGNTYYADSVKGRFTVSRDNAKHTVYLQMNSLKPEDTAVYYCAAASFAPGSRGYDYWGQGTQVTVSS >180F5 Notch-1 346EVQLVESGGGLVQPGGSLRLSCAASGRTSSPIAMGWFRQAPGKEREFVSGITWSGAYTHYANSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCTTATNSTTGYDYWGQGTQVTVSS >180F10 Notch-1 347EVQLVESGGDLVQPGGSLRLSCAASATVFSNSDMGWSRQAPGKERELVASITTDGNTLYADAAKGRFAIYRDNSKNTVYLEMNSLKPDDTAVYYCRVVDLLRGKLYWGQGTQVTVSS >181G4 Notch-2 348EVQLVESGGGLVQAGGSLRLSCAASGSIFSITEMDWYRQAPGKQREWVAGETSDGSTNYADSVKGRFTISRDNANNAVYLQMNRLKPEDTAVYHCAASLRNSGSNVEGRYWGQGTQVTVSS >181B4 Notch-2 349EVQLVESGGGLVQPGGSLRLSCAASGRINSMNWYHQAPGKEREWVASITSSGTAIYADSVKGRITISRDNAKNTVYLQMNSLKTEDTGVYYCAANLQNARGSYYGQGTQVTVSS >181D4 Notch-2 350EVQLVESGGGLVQPGGSLRLSCAASGFTFGDYAMNWVRQAPGKGLEWVSSISAGGYSTTYADSVKGRFTISRDNAKNTLYLQMNSLNPEDTAVYYCARGDWRYGSRGQGTQVTVSS >181E4 Notch-2 351EVQLVESGGGLVQAGGSLRLSCAASGSMSSINAMRWYRQASGKQREPVAEITSEGTIIYADSVKGRFTTSRDNAKNTVYLQMNSLKPEDTGVYYCAADDGARGSYYGQGTQVTVSS >181F4 Notch-2 352EVQLVESGGALVQAGGSLRLSCVASGSSFSINDMDWYRQAPGKTREWVAGINEYGGRNYANSVKDRFTISTDNAKNTVYLQMNSLKPEDTGVYYCAATLAKGGGRYWGQGTPVTVSS >181H4 Notch-2 353EVKLVESGGGLVQPGGSLRLSCAASGSIFRFNGVDWYRQAPGAEREWVAGFGSGGTTNYADSVKGRFIVSRDNAENTVFLQMNSLKPEDSAVYFCAASIEGVSGRYYGQGTQVTVSS >181A5 Notch-2 354EVQLVESGGGLVQAGGSLRLSCVVSGSILSINTMDWYRQAPGNEREWVGGITDGGRSNYADSVKDRFTIYRANAKNTVYLQMNSLKPEDTAVYYCAADLRGGIATTGRYWGQGTQVTVSS >181C10 Notch-2 355EVQLVESGGGLVQAGGSLRLSCAASGMTTIGPMGWYRQGPGKQRELVARITGGGSTNYVDSAKGRFTISRDNAKNAVYLQMNNLKLDDTAVYYCNAYGSGSDYLPIDYWGQGTQVTVSS >181C11 Notch-2 356EVQLVESGGGLVQAGGSLRLSCAASGMTTIGPMGWYRQGPGKQRELVARITGGGSTNYVDSAKGRFTISRDNAKNMGYLQMNNLKLDDTAIYYCYAYGSGSDYLPTDYWGQGTQVTVSS >181D11 Notch-2 357EVQLVESGGGLVQAGGSLRLSCAASGMTTIGPMGWYRQGPGKQRELVARITGGGSTNYVDSAKGRFTISRHNAKNMVYLQMNNLKLDDTAVYYCNAYGSGSDYLPMDYWGQGTQVTVSS (“Clone name” (first column)and “SEQ ID NO: XXX” are also used to refer to the correspondingpreferred single variable domain sequence itself and all three terms canbe used interchangeably, e.g. terms: “178C4”, “SEQ ID NO: 252” and“EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAMAWFRQAPGKEREFVAAISWSGDSTSYADSVKGRFTISRHNAENTVFLQMNSLKPEDTAVYYCAADRRVRVHGSWYGSEYWGQGTQVTVSS” can be used interchangeably)

TABLE A-1B Preferred combinations of CDR sequences, preferredcombinations of framework sequences, and preferred combinations offramework and CDR sequences. Pre- ferred combi- nations FR1 ID CDR1 IDFR2 ID CDR2 ID FR3 ID CDR3 ID FR4 ID 1 EVQLVESG 126 IYDMG 155 WFRQAPGKE184 SIHWRGRGTN 213 RFTISKDNA 242 TRLIVYTPTGFR 271 WGQGAQ 300 GGLVQAGGREFVS YTDSVEG KNMVYLQM QYFD VTVSS SLRLSCIAS NSLKPEDTA GDTSE VYYCAA 2EVQLVESG 127 SDVMG 156 WFRRAPGKE 185 SITTTGNEYYE 214 RFTVSRDIA 243RLLGSTIRSHEY 272 WGQGTQ 301 GGLVQAGG REFVA DSLKG ENTMYLEMT RY VTVSSSLRLSCAAS NLKPEDTAE GxxFx YSCAG 3 EVQLVESG 128 IYDMG 157 WFRQAPGKE 186SIHWGGRGTN 215 RFTISKDNA 244 TRLIVYTPTGFR 273 WGQGTQ 302 GGLVQAGG REFVSYTDSVKG KNMVHLQM QYFD VTVSS SLRLSCVAS NSLKPEDTA GDTSE VYYCAA 4 EVQLVESG129 IYDMG 158 WFRQAPGKE 187 SIHWGGRGTN 216 RFTISKDNA 245 TRLIVYTPTGFR274 WGQGTQ 303 GGLVQAGG REFVS YTDSVKG KNMVYLQM QYFD VTVSS SLRLSCVASNSLKPEDTA GDTSE VYYCAA 5 EVQLVESG 130 GFLLG 159 WYRQPPGKQ 188 QTTDGGRTNY217 RFTISRDNA 246 FPLTS 275 WGQGTQ 304 GGLVQAGG RELIA GDSVKG KNTWYLQMVTVSS SLRLSCAAS DSLKPEDTG GSDFS VYYCNT 6 EVQLVESG 131 INDMG 160WYRQAPGKT 189 TITRSGVLNYT 218 RFTVSRDDA 247 NIVIRSSGYFNRY 276 WGQGTLV305 GGLVQSGG REMVA DSVKG KNTVYLQMS TVSS SLSLSCAAS SLKPEDTAV GSVLR YSCFA7 EVQLVESG 132 DYAIG 161 WFRQAPGKE 190 CMRGRDGST 219 RFTISSDNA 248HTDFACYIGYYS 277 WGQGTQ 306 GGLVQAGG REGVS YYADSVNG KNTVYLQMN DYDPHDYVTVSS SLRLSCAAS SLKPEDTAV GFTFD YYCAA 8 EVQLVESG 133 TAAMG 162 WYRQAPGKS191 RMFSDGRTIY 220 RFTISRDNA 249 LQFGAVY 278 WGQGTQ 307 GGLVQPGG RVLVAAESVKR KNTVYLQMN VTVSS SLRLSCATS SLEPEDTAV GVIFS YYCNA 9 EVQLVKSG 134ANLIG 163 WYRQAPGKQ 192 FITSGGAINYA 221 RFTISRDDA 250 RQLGNVY 279 WAQGTQ308 GGLVQPGG REGVA DSVKG KNTVYLQMN VTVSS SLRLSCAAS SLKPEDTAIY GSIVS YCNL10 EVQLVESG 135 TAAMG 164 WYRQAPGKS 193 RMFSDGRTIY 222 RFTISRDNA 251LQFGAVY 280 WGQGTQ 309 GGLVQPGG RVLVA AESVKR KNTVYLQMN VTVSS SLRLSCATSSLEPEDTAV GVIFS YYCNA 11 EVQLVESG 136 PIAMG 165 WFRQAPGKE 194 GITWSGAYTH223 RFTISRDSA 252 ATNSTTGYDY 281 WGQGTQ 310 GGLVQPGG REFVS YANSVKGKNTVYLQMN VTVSS SLRLSCAAS SLKPEDTAV GRTSS YYCTT 12 EVQLVESG 137 NNDMG166 WSRQAPGKE 195 SFTSGGNTVY 224 RFTISRDNS 253 VDLLRGKLY 282 WGQGTQ 311GDLVQPGG RELVA ADAAKG KNTVYLEMN VTVSS SLRLSCAAS SLKPDDTAV GTVFS YYCRV 13EVQLVESG 138 NNDMG 167 WSRQAPGKE 196 SFTSGGNTVY 225 RFTISRDNS 254VDLLRGKLY 283 WGQGTQ 312 GDLVQPGG RELVA ADAAKG KNTVYLEMN VTVSS SLRLSCAASSLKPDDTAV GTVFS YYCRV 14 EVQLVESG 139 ISDMG 168 WSRQAPGKE 197SISSDNYTVYA 226 RFTISRDNS 255 VDPLRGKLY 284 WGQGTQ 313 GDLVQPGG RELVADAAKG KNTVYLEMN VTVSS SLRLSCAAS SLKPDDTAV RTVFS YYCRV 15 EVQLVESG 140INAVA 169 WYRQAPGKE 198 LIVGEAITRYT 227 RFTISRDNA 256 VDPLRGKLY 285WGQGTQ 314 GGLVQAGG RDWVA DSVSG KNTVYLEMN VTVSS SLRLSCAAS SLKPDDTAVGGIFG YYCRV 16 EVQLVESG 141 INDMG 170 WSRQAPGKG 199 SISSEGTTIYA 228RFTISRDNS 257 VDPLRGKLY 286 WGQGTQ 315 GDLVQPGG RELVA DAAKG KNTVYLEMNVTVSS SLRLSCTTS SLKPDDTAV GTVFS YYCRV 17 EVQLVESG 142 SYTMG 171WVRQAPGKE 200 SIWRSGGNTY 229 RFTVSRDNA 258 ASFAPGSRGYDY 287 WGQGTQ 316GGLVQAGD REAVA YADSVKG KHTVYLQMN VTVSS SLRLSCAVS SLKPEDTAV GGSFS YYCAA18 EVQLVESG 143 PIAMG 172 WFRQAPGKE 201 GITWSGAYTH 230 RFTISRDSA 259ATNSTTGYDY 288 WGQGTQ 317 GGLVQPGG REFVS YANSVKG KNTVYLQMN VTVSSSLRLACAAS SLKPEDTAV GRTSS YYCTT 19 EVQLVESG 144 NSDMG 173 WSRQAPGKE 202SITTDGNTLYA 231 RFAIYRDNS 260 VDLLRGKLY 289 WGTQGTQ 318 GDLVQPGG RELVADAAKG KNTVYLEMN VTVSS SLRLSCAAS SLKPDDTAV ATVFS YYCRV 20 EVQLVESG 145ITEMD 174 WYRQAPGKQ 203 GETSDGSTNY 232 RFTISRDNA 261 SLRNSGSNVEG 290WGQGTQ 319 GGLVQAGG REWVA ADSVKG NNAVYLQM RY VTVSS SLRLSCAAS NRLKPEDTAGSIFS VYHCAA 21 EVQLVESG 146 MN 175 WYHQAPGKE 204 SITSSGTAIYA 233RITISRDNAK 262 NLQNARGSY 291 YGQGTQV 320 GGLVQPGG REWVA DSVKG NTVYLQMNSTVSS SLRLSCAAS LKTEDTGVY GRINS YCAA 22 EVQLVESG 147 DYAMN 176 WVRQAPGKG205 SISAGGYSTTY 234 RFTISRDNA 263 GDWRYGS 292 RGQGTQV 321 GGLVQPGG LEWVSADSVKG KNTLYLQMN TVSS SLRLSCAAS SLNPEDTAV GFTFG YYCAR 23 EVQLVESG 148INAMR 177 WYRQASGKQ 206 EITSEGTIIYAD 235 RFTTSRDNA 264 DDGARGSY 293YGQGTQV 322 GGLVQAGG REPVA SVKG KNTVYLQMN TVSS SLRLSCAAS SLKPEDTGV GSMSSYYCAA 24 EVQLVESG 149 INDMD 178 WYRQAPGKT 207 GINEYGGRNY 236 RFTISTDNAK265 TLAKGGGRY 294 WGQGTP 323 GALVQAGG REWVA ANSVKD NTVYLQMNS VTVSSSLRLSCVAS LKPEDTGVY GSSFS YCAA 25 EVKLVESGG 150 FNGVD 179 WYRQAPGAE 208GFGSGGTTNY 237 RFIVSRDNA 266 SIEGVSGRY 295 YGQGTQV 324 GLVQPGGS REWVAADSVKG ENTVFLQMN TVSS LRLSCAASG SLKPEDSAV SIFR YFCAA 26 EVQLVESG 151INTMD 180 WYRQAPGNE 209 GITDGGRSNY 238 RFTIYRANA 267 DLRGGIATTGRY 296WGQGTQ 325 GGLVQAGG REWVG ADSVKD KNTVYLQMN VTVSS SLRLSCVVS SLKPEDTAVGSILS YYCAA 27 EVQLVESG 152 GPMG 181 WYRQGPGKQ 210 RITGGSTNY 239RFTISRDNA 268 YGSGSDYLPIDY 297 WGQGTQ 326 GGLVQAGG RELVA VDSAKGKNAVYLQMN VTVSS SLRLSCAAS NLKLDDTAV GMTTI YYCNA 28 EVQLVESG 153 GPMG 182WYRQGPGKQ 211 RITGGGSTNY 240 RFTISRDNA 269 YGSGSDYLPTDY 298 WGQGTQ 327GGLVQAGG RELVA VDSAKG KNMGYLQM VTVSS SLRLSCAAS NNLKLDDTAI GMTTI YYCYA 29EVQLVESG 154 GPMG 183 WYRQGPGKQ 212 RITGGGSTNY 241 RFTISRHNA 270YGSGSDYLPMDY 299 WGQGTQ 328 GGLVQAGG RELVA VDSAKG KNMVYLQM VTVSSSLRLSCAAS NNLKLDDTA GMTTI VYYCNA (“ID” refers to the SEQ ID NO and isreferenced e.g. below whereas “ID”/“ID SEQ ID NO” number on the right ofthe sequence refers to corresponding sequence)

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 present is suitably chosen from the group consisting of theCDR1, CDR2 and CDR3, respectively, listed in Table A-1B; or from thegroup of CDR1, CDR2 and CDR3, respectively, that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% “sequence identity” (as defined herein) with atleast one of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B; and/or from the group consisting of the CDR1, CDR2 and CDR3,respectively, that have 3, 2 or only 1 “amino acid difference(s)” (asdefined herein) with at least one of the CDR1, CDR2 and CDR3,respectively, listed in Table A-1B.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 (i.e. as defined herein), aCDR2 sequence is chosen from suitable CDR2 (i.e. as defined herein), anda CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as definedherein), respectively. More in particular, the CDR sequences arepreferably chosen such that the Nanobodies of the invention bind tomembers of the Notch signalling pathway with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1B or from the group of CDR3 that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of the CDR3listed in Table A-1B; and/or from the group consisting of the CDR3 thathave 3, 2 or only 1 amino acid difference(s) with at least one of theCDR3 listed in Table A-1B.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B or from the group consisting of CDR1, CDR2 and CDR3, respectively,that have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B; and/or from the group consisting of the CDR1, CDR2 and CDR3,respectively, that have 3, 2 or only 1 “amino acid difference(s)” withat least one of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1B or from the group of CDR3 that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of the CDR3listed in Table A-1B, respectively; and at least one of the CDR1 andCDR2 present is suitably chosen from the group consisting of the CDR1and 180-197, respectively, listed in Table A-1B or from the group ofCDR1 and CDR2, respectively, that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the CDR1 and CDR2, respectively,listed in Table A-1B; and/or from the group consisting of the CDR1 andCDR2, respectively, that have 3, 2 or only 1 amino acid difference(s)with at least one of the CDR1 and CDR2, respectively, listed in TableA-1B.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 present are suitably chosen from the group consisting ofthe CDR1, CDR2 and CDR3, respectively, listed in Table A-1B or from thegroup of CDR1, CDR2 and CDR3, respectively, that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of the CDR1,CDR2 and CDR3, respectively, listed in Table A-1B; and/or from the groupconsisting of the CDR1, CDR2 and CDR3, respectively, that have 3, 2 oronly 1 amino acid difference(s) with at least one of the CDR1, CDR2 andCDR3, respectively, listed in Table A-1B.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 present is suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B. Preferably, in this aspect, at least one or preferably both of theother two CDR sequences present are suitably chosen from CDR sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the corresponding CDR sequences, respectively, listed inTable A-1B; and/or from the group consisting of the CDR sequences thathave 3, 2 or only 1 amino acid difference(s) with at least one of thecorresponding sequences, respectively, listed in Table A-1B.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1B. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 present are suitably chosen fromthe groups of CDR1 and CDR2, respectively, that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with the CDR1 and CDR2,respectively, listed in Table A-1B; and/or from the group consisting ofthe CDR1 and CDR2, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2, respectively,listed in Table A-1B.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B. Preferably, in this aspect, the remaining CDR sequence present issuitably chosen from the group of CDR sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of thecorresponding CDR sequences listed in Table A-1B; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with at least one of the corresponding sequences listed inTable A-1B.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the 216-233listed in Table A-1B, and either the CDR1 sequence or the CDR2 sequenceis suitably chosen from the group consisting of the CDR1 and CDR2,respectively, listed in Table A-1B. Preferably, in this aspect, theremaining CDR sequence present is suitably chosen from the group of CDRsequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable A-1B; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table A-1B.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3, respectively, listed in TableA-1B.

Also, generally, the combinations of CDR's listed in Table A-1B (i.e.those mentioned on the same line in Table A-1B) are preferred. Thus, itis generally preferred that, when a CDR in a Nanobody of the inventionis a CDR sequence mentioned in Table A-1B or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table A-1B; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table A-1B, that at leastone and preferably both of the other CDR's are suitably chosen from theCDR sequences that belong to the same combination in Table A-1B (i.e.mentioned on the same line in Table A-1B) or are suitably chosen fromthe group of CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with the CDR sequence(s) belonging to the samecombination and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the CDR sequence(s)belonging to the same combination. The other preferences indicated inthe above paragraphs also apply to the combinations of CDR's mentionedin Table A-1B.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 mentioned in Table A-1B, a CDR2 sequencethat has 3, 2 or 1 amino acid difference with one of the CDR2 mentionedin Table A-1B (but belonging to a different combination), and a CDR3sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 mentioned in Table A-1B; a CDR2 sequence that has 3, 2 or 1 aminoacid difference with one of the CDR2 mentioned in Table A-1B (butbelonging to a different combination); and a CDR3 sequence that has morethan 80% sequence identity with one of the CDR3 mentioned in Table A-1B(but belonging to a different combination); or (2) a CDR1 sequence thathas more than 80% sequence identity with one of the CDR1 mentioned inTable A-1B; a CDR2 sequence, and one of the CDR3 listed in Table A-1B;or (3) a CDR1 sequence; a CDR2 sequence that has more than 80% sequenceidentity with one of the CDR2 sequence listed in Table A-1B; and a CDR3sequence that has 3, 2 or 1 amino acid differences with the CDR3sequence mentioned in Table A-1B that belongs to the same combination asthe CDR2 sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 mentioned in Table A-1B; a CDR2 sequence that has3, 2 or 1 amino acid difference with the CDR2 sequence mentioned inTable A-1B that belongs to the same combination; and a CDR3 sequencethat has more than 80% sequence identity with the CDR3 sequencementioned in Table A-1B that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table A-1B and a CDR3 sequence listed inTable A-1B (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 mentioned in Table A-1B; the CDR2 sequence listedin Table A-1B that belongs to the same combination; and a CDR3 sequencementioned in Table A-1B that belongs to a different combination; or (2)a CDR1 sequence mentioned in Table A-1B; a CDR2 sequence that has 3, 2or 1 amino acid differences with the CDR2 sequence mentioned in TableA-1B that belongs to the same combination; and a CDR3 sequence that hasmore than 80% sequence identity with the CDR3 sequence listed in TableA-1B that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table A-1B, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable A-1B that belongs to the same combination; and the CDR3 sequencementioned in Table A-1B that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 andCDR3 present are suitably chosen from one of the combinations of CDR1,CDR2 and CDR3, respectively, listed in Table A-1B.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the single variable domains of SEQ ID NO's:[255-269].

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such Nanobodies may be naturally occurring Nanobodies (from any suitablespecies), naturally occurring VHH sequences (i.e. from a suitablespecies of Camelid) or synthetic or semi-synthetic single variabledomains or Nanobodies, including but not limited to partially humanizedNanobodies or VHH sequences, fully humanized Nanobodies or VHHsequences, camelized heavy chain variable domain sequences, as well asNanobodies that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the single variable domains of SEQ ID NO's:[255-269]. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said Nanobody and one or more of the sequencesof SEQ ID NO's: [255-269], in which the amino acid residues that formthe framework regions are disregarded. Such Nanobodies can be as furtherdescribed herein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: [255-269] or from the group consisting offrom single variable domains that have more than 80%, preferably morethan 90%, more preferably more than 95%, such as 99% or more sequenceidentity (as defined herein) with at least one of the single variabledomains of SEQ ID NO's: [255-269].

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: [255-269], thatcomprise, compared to the corresponding native V_(HH) sequence, at leastone humanizing substitution (as defined herein), and in particular atleast one humanizing substitution in at least one of its frameworksequences (as defined herein).

The single variable domains of the invention comprise or essentiallyconsist of at least one Nanobody of the invention. Some preferred, butnon-limiting examples of single variable domains of the invention aregiven in SEQ ID NO's: [255-269].

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the single variable domains describedherein. Thus, single variable domains that comprise or essentiallyconsist of one or more “preferred” Nanobodies of the invention willgenerally be preferred, and single variable domains that comprise oressentially consist of one or more “more preferred” Nanobodies of theinvention will generally be more preferred, etc.

Generally, proteins or single variable domains that comprise oressentially consist of a single Nanobody (such as a single Nanobody ofthe invention) will be referred to herein as “monovalent” proteins orsingle variable domains or as “monovalent constructs”. Proteins andsingle variable domains that comprise or essentially consist of two ormore Nanobodies (such as at least two Nanobodies of the invention or atleast one Nanobody of the invention and at least one other Nanobody)will be referred to herein as “multivalent” proteins or single variabledomains or as “multivalent constructs”, and these may provide certainadvantages compared to the corresponding monovalent Nanobodies of theinvention. Some non-limiting examples of such multivalent constructswill become clear from the further description herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for members for the Notch signalling pathway. Suchmultivalent constructs will be clear to the skilled person based on thedisclosure herein.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one Nanobodyof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a Nanobody. Such proteins or single variable domainsare also referred to herein as “multispecific” proteins or singlevariable domains or as ‘multispecific constructs”, and these may providecertain advantages compared to the corresponding monovalent Nanobodiesof the invention (as will become clear from the further discussionherein of some preferred, but-nonlimiting multispecific constructs).Such multispecific constructs will be clear to the skilled person basedon the disclosure herein.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such of such fusion constructs will becomeclear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or other singlevariable domains may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andsingle variable domains will become clear to the skilled person based onthe further disclosure herein, and for example comprise Nanobodiessequences or single variable domains of the invention that have beenchemically modified to increase the half-life thereof (for example, bymeans of pegylation); single variable domains of the invention thatcomprise at least one additional binding site for binding to a serumprotein (such as serum albumin, see for example EP 0 368 684 B1, page4); or single variable domains of the invention that comprise at leastone Nanobody of the invention that is linked to at least one moiety (andin particular at least one amino acid sequence) that increases thehalf-life of the Nanobody of the invention. Examples of single variabledomains of the invention that comprise such half-life extending moietiesor single variable domains will become clear to the skilled person basedon the further disclosure herein; and for example include, withoutlimitation, single variable domains in which the one or more Nanobodiesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as serum albumin or suitable fragments thereof)or to one or more binding units that can bind to serum proteins (suchas, for example, Nanobodies or (single) domain antibodies that can bindto serum proteins such as serum albumin, serum immunoglobulins such asIgG, or transferrin); single variable domains in which a Nanobody of theinvention is linked to an Fc portion (such as a human Fc) or a suitablepart or fragment thereof; or single variable domains in which the one ormore Nanobodies of the invention are suitable linked to one or moresmall proteins or peptides that can bind to serum proteins (such as,without limitation, the proteins and peptides described in WO 91/01743,WO 01/45746, WO 02/076489 and to the US provisional application ofAblynx N.V. entitled “Peptides capable of binding to serum proteins” ofAblynx N.V. filed on Dec. 5, 2006 (see also PCT/EP/2007/063348).

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or single variable domains may contain one or moreadditional groups, residues, moieties or binding units, such as one ormore further in particular one or more additional Nanobodies (i.e. notdirected against members for the Notch signalling pathway), so as toprovide a tri- of multispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs orsingle variable domains comprising the same) with increased half-lifepreferably have a half-life that is at least 1.5 times, preferably atleast 2 times, such as at least 5 times, for example at least 10 timesor more than 20 times, greater than the half-life of the correspondingamino acid sequence of the invention per se. For example, theNanobodies, compounds, constructs or single variable domains of theinvention with increased half-life may have a half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or single variable domains of theinvention exhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orsingle variable domains of the invention may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) single variable domainsthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more single variable domainsthat allow the resulting single variable domains of the invention tocross the blood brain barrier may be one or more (such as two andpreferably one) Nanobodies, such as the Nanobodies described in WO02/057445, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQID NO: 190 of WO 06/040154) are preferred examples.

In particular, single variable domains comprising one or more Nanobodiesof the invention are preferably such that they:

-   -   bind to members of the Notch signalling pathway with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to members of the Notch signalling pathway with a        k_(on)-rate of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,        preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably        between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹        and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to members of the Notch signalling pathway with a k_(off)        rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a        near irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to members of theNotch signalling pathway with an affinity less than 500 nM, preferablyless than 200 nM, more preferably less than 10 nM, such as less than 500pM. In this respect, it will be clear to the skilled person that apolypeptide that contains two or more Nanobodies of the invention maybind to members of the Notch signalling pathway with an increasedavidity, compared to a polypeptide that contains only one amino acidsequence of the invention.

Some preferred IC₅₀ values for binding of the single variable domains orsingle variable domains of the invention to members of the Notchsignalling pathway will become clear from the further description andexamples herein.

Other single variable domains according to this preferred aspect of theinvention may for example be chosen from the group consisting of singlevariable domains that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the single variable domains of SEQID NO's: [255-269], in which the Nanobodies comprised within said singlevariable domains are preferably as further defined herein.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as a Nanobody of theinvention) or a polypeptide of the invention comprising the same. Again,as generally described herein for the nucleic acids of the invention,such a nucleic acid may be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence (suchas a Nanobody) of the invention and/or a polypeptide of the inventioncomprising the same; and/or that contains a nucleic acid of theinvention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one amino acid sequence of theinvention, at least one polypeptide of the invention and/or at least onenucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, compounds, constructs, single variable domains,nucleic acids, host cells, products and compositions described herein.Some preferred but non-limiting examples of such methods will becomeclear from the further description herein.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, single variable domains, nucleicacids, host cells, products and compositions described herein, as wellas to methods for the prevention and/or treatment for diseases anddisorders associated with members for the Notch signalling pathway. Somepreferred but non-limiting applications and uses will become clear fromthe further description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained: (1) by isolating the V_(HH) domain of a naturally occurringheavy chain antibody; (2) by expression of a nucleotide sequenceencoding a naturally occurring V_(HH) domain; (3) by “humanization” (asdescribed herein) of a naturally occurring V_(HH) domain or byexpression of a nucleic acid encoding a such humanized V_(HH) domain;(4) by “camelization” (as described herein) of a naturally occurringV_(H) domain from any animal species, and in particular a from speciesof mammal, such as from a human being, or by expression of a nucleicacid encoding such a camelized V_(H) domain; (5) by “camelisation” of a“domain antibody” or “Dab” as described by Ward et al (supra), or byexpression of a nucleic acid encoding such a camelized V_(H) domain; (6)by using synthetic or semi-synthetic techniques for preparing proteins,single variable domains or other single variable domains known per se;(7) by preparing a nucleic acid encoding a Nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing. Suitable methods and techniques for performing theforegoing will be clear to the skilled person based on the disclosureherein and for example include the methods and techniques described inmore detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against members forthe Notch signalling pathway. As further described herein, such VHHsequences can generally be generated or obtained by suitably immunizinga species of Camelid with members of the Notch signalling pathway (i.e.so as to raise an immune response and/or heavy chain antibodies directedagainst members for the Notch signalling pathway), by obtaining asuitable biological sample from said Camelid (such as a blood sample,serum sample or sample of B-cells), and by generating VHH sequencesdirected against members for the Notch signalling pathway, starting fromsaid sample, using any suitable technique known per se. Such techniqueswill be clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against membersfor the Notch signalling pathway, can be obtained from naïve librariesof Camelid VHH sequences, for example by screening such a library usingmembers for the Notch signalling pathway, or at least one part,fragment, antigenic determinant or epitope thereof using one or morescreening techniques known per se. Such libraries and techniques are forexample described in WO 99/37681, WO 01/90190, WO 03/025020 and WO03/035694. Alternatively, improved synthetic or semi-synthetic librariesderived from naïve V_(HH) libraries may be used, such as V_(HH)libraries obtained from naïve V_(HH) libraries by techniques such asrandom mutagenesis and/or CDR shuffling, as for example described in WO00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against members for the Notchsignalling pathway. In one aspect, said method at least comprises thesteps of:

-   a) providing a set, collection or library of Nanobody sequences; and-   b) screening said set, collection or library of Nanobody sequences    for Nanobody sequences that can bind to and/or have affinity for    members for the Notch signalling pathway;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for members for the Notch signalling pathway.

In such a method, the set, collection or library of Nanobody sequencesmay be a naïve set, collection or library of Nanobody sequences; asynthetic or semi-synthetic set, collection or library of Nanobodysequences; and/or a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of VHH sequences, that have been derived from a species ofCamelid that has been suitably immunized with members of the Notchsignalling pathway or with a suitable antigenic determinant basedthereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of Nanobody or VHHsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) Nanobody sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to WO 03/054016 and tothe review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116(2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for members for the Notch signalling pathway; and (ii)    cells that express heavy chain antibodies, in which substeps (i)    and (ii) can be performed essentially as a single screening step or    in any suitable order as two separate screening steps, so as to    provide at least one cell that expresses a heavy chain antibody that    can bind to and/or has affinity for members for the Notch signalling    pathway;    and-   c) either (i) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present in    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with members of the Notch signalling pathway ora suitable antigenic determinant based thereon or derived therefrom,such as an antigenic part, fragment, region, domain, loop or otherepitope thereof. In one particular aspect, said antigenic determinantmay be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone™” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against members of the Notch signalling pathway may comprise atleast the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or Nanobody sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a Nanobody sequence that can bind to and/or has affinity    for members for the Notch signalling pathway;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orVHH sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofsingle variable domains may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or VHH sequencesderived from a Camelid that has been suitably immunized with members ofthe Notch signalling pathway or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding single variable domains will be clear to the person skilled inthe art, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of single variable domains may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired single variabledomains of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining VHH sequences or Nanobody sequencesdirected against members of the Notch signalling pathway involvessuitably immunizing a transgenic mammal that is capable of expressingheavy chain antibodies (i.e. so as to raise an immune response and/orheavy chain antibodies directed against members for the Notch signallingpathway), obtaining a suitable biological sample from said transgenicmammal that contains (nucleic acid sequences encoding) said VHHsequences or Nanobody sequences (such as a blood sample, serum sample orsample of B-cells), and then generating VHH sequences directed againstmembers for the Notch signalling pathway, starting from said sample,using any suitable technique known per se (such as any of the methodsdescribed herein or a hybridoma technique). For example, for thispurpose, the heavy chain antibody-expressing mice and the furthermethods and techniques described in WO 02/085945, WO 04/049794 and WO06/008548 and Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10;103(41):15130-5 can be used. For example, such heavy chain antibodyexpressing mice can express heavy chain antibodies with any suitable(single) variable domain, such as (single) variable domains from naturalsources (e.g. human (single) variable domains, Camelid (single) variabledomains or shark (single) variable domains), as well as for examplesynthetic or semi-synthetic (single) variable domains.

The invention also relates to the VHH sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said V_(HH) sequence or Nanobody sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized Nanobodies of the invention can be obtained inany suitable manner known per se (i.e. as indicated under points (1)-(8)above) and thus are not strictly limited to single variable domains thathave been obtained using a polypeptide that comprises a naturallyoccurring V_(HH) domain as a starting material.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996), supra). Preferably, the V_(H) sequence thatis used as a starting material or starting point for generating ordesigning the camelized Nanobody is preferably a V_(H) sequence from amammal, more preferably the V_(H) sequence of a human being, such as aV_(H)3 sequence. However, it should be noted that such camelizedNanobodies of the invention can be obtained in any suitable manner knownper se (i.e. as indicated under points (1)-(8) above) and thus are notstrictly limited to single variable domains that have been obtainedusing a polypeptide that comprises a naturally occurring V_(H) domain asa starting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” Nanobody of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired Nanobody of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for peptide synthesis known per se. Also, based on the aminoacid sequence or nucleotide sequence of a naturally occurring V_(HH)domain or V_(H) domain, respectively, a nucleotide sequence encoding thedesired humanized or camelized Nanobody of the invention, respectively,can be designed and then synthesized de novo using techniques fornucleic acid synthesis known per se, after which the nucleic acid thusobtained can be expressed in a manner known per se, so as to provide thedesired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably VHH sequences, will beclear from the skilled person, and may for example comprise combiningone or more parts of one or more naturally occurring V_(H) sequences(such as one or more FR sequences and/or CDR sequences), one or moreparts of one or more naturally occurring VHH sequences (such as one ormore FR sequences or CDR sequences), and/or one or more synthetic orsemi-synthetic sequences, in a suitable manner, so as to provide aNanobody of the invention or a nucleotide sequence or nucleic acidencoding the same (which may then be suitably expressed). Nucleotidesequences encoding framework sequences of VHH sequences or Nanobodieswill be clear to the skilled person based on the disclosure hereinand/or the further prior art cited herein (and/or may alternatively beobtained by PCR starting from the nucleotide sequences obtained usingthe methods described herein) and may be suitably combined withnucleotide sequences that encode the desired CDR's (for example, by PCRassembly using overlapping primers), so as to provide a nucleic acidencoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized bythe presence of one or more “Hallmark residues” (as described herein) inone or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody against members of the Notch signallingpathway according to the invention may have the structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q; or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q; and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as    described herein) and the amino acid residue at position 108 is Q;    or in which:-   ii) the amino acid residues at positions 43-46 according to the    Kabat numbering form the sequence KERE or KQRE (or a KERE-like    sequence as described) and the amino acid residue at position 108 is    Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   i) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table A-3 below. More generally, and    without limitation, Nanobodies belonging to the GLEW-group can be    defined as Nanobodies with a G at position 44 and/or with a W at    position 47, in which position 46 is usually E and in which    preferably position 45 is not a charged amino acid residue and not    cysteine;-   ii) The “KERE-group”: Nanobodies with the amino acid sequence KERE    or KQRE (or another KERE-like sequence) at positions 43-46 according    to the Kabat numbering and Q or L at position 108 according to the    Kabat numbering. As further described herein, Nanobodies within this    group usually have a F at position 37, an L or F at position 47; and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. More generally, and without limitation, Nanobodies    belonging to the KERE-group can be defined as Nanobodies with a K, Q    or R at position 44 (usually K) in which position 45 is a charged    amino acid residue or cysteine, and position 47 is as further    defined herein;-   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at    position 103. These Nanobodies can have either the amino acid    sequence GLEW at positions 44-47 according to the Kabat numbering or    the amino acid sequence KERE or KQRE at positions 43-46 according to    the Kabat numbering, the latter most preferably in combination with    an F at position 37 and an L or an F at position 47 (as defined for    the KERE-group); and can have Q or L at position 108 according to    the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred toabove describe and apply to Nanobodies in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNanobodies may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized Nanobodies of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,Sresidues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2). Such substitutions include, butare not limited to, the GLEW-like sequences mentioned in Table A-3below; as well as the substitutions that are described in theInternational Application WO 00/29004 for so-called “microbodies”, e.g.so as to obtain a Nanobody with Q at position 108 in combination withKLEW at positions 44-47. Other possible substitutions at these positionswill be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residueat position 83 is chosen from the group consisting of L, M, S, V and W;and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acidresidue at position 83 is chosen from the group consisting of R, K, N,E, G, I, T and Q; and is most preferably either K or E (for Nanobodiescorresponding to naturally occurring V_(HH) domains) or R (for“humanized” Nanobodies, as described herein). The amino acid residue atposition 84 is chosen from the group consisting of P, A, R, S, D T, andV in one aspect, and is most preferably P (for Nanobodies correspondingto naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies,as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the aminoacid residue at position 104 is chosen from the group consisting of Gand D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table A-3.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table A-4. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; L, M, S, V, W; preferably L predominantly L  37 V, I,F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾,E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾or E⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; preferablyW⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Qor T; preferably K or R; most preferably K  84 A, T, D; P⁽⁵⁾, A, L, R,S, T, D, V; preferably P predominantly A 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S;preferably W 104 G G or D; preferably G 108 L, M or T; Q, L⁽⁷⁾ or R;preferably Q or L⁽⁷⁾ predominantly L Notes: ⁽¹⁾In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46.⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG atpositions 43-47. Alternatively, also sequences such as TERE (for exampleTEREL), KECE (for example KECEL or KECER), RERE (for example REREG),QERE (for example QEREG), KGRE (for example KGREG), KDRE (for exampleKDREV) are possible. Some other possible, but less preferred sequencesinclude for example DECKL and NVCEL. ⁽⁴⁾With both GLEW at positions44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP or EP atpositions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾In particular,but not exclusively, in combination with GLEW at positions 44-47.⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position 108 isalways Q in (non-humanized) V_(HH) sequences that also contain a W atposition 103. The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW,ELEW, GPEW, EWLP, GPER, GLER and ELEW.

TABLE A-4 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. For humanization of thesecombinations, reference is made to the specification. 11 37 44 45 47 8384 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R LK P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q LF L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group LV G L W K S W G Q M V G L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5to A-8 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1.)

In Tables A-5-A-8, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 1118 V_(HH) sequences(data kindly provided by David Lutje Hulsing and Prof. Theo Verrips ofUtrecht University). The values for the V_(HH) entropy and the V_(HH)variability provide a measure for the variability and degree ofconservation of amino acid residues between the 1118 V_(HH) sequencesanalyzed: low values (i.e. <1, such as <0.5) indicate that an amino acidresidue is highly conserved between the V_(HH) sequences (i.e. littlevariability). For example, the G at position 8 and the G at position 9have values for the V_(HH) entropy of 0.1 and 0 respectively, indicatingthat these residues are highly conserved and have little variability(and in case of position 9 is G in all 1118 sequences analysed), whereasfor residues that form part of the CDR's generally values of 1.5 or moreare found (data not shown). Note that (1) the amino acid residues listedin the second column of Tables A-5-A-8 are based on a bigger sample thanthe 1118 V_(HH) sequences that were analysed for determining the V_(HH)entropy and V_(HH) variability referred to in the last two columns; and(2) the data represented below support the hypothesis that the aminoacid residues at positions 27-30 and maybe even also at positions 93 and94 already form part of the CDR's (although the invention is not limitedto any specific hypothesis or explanation, and as mentioned above,herein the numbering according to Kabat is used). For a generalexplanation of sequence entropy, sequence variability and themethodology for determining the same, see Oliveira et al., PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q Q, A, E — — 2V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D,Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V,A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 515 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 322 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 625 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R,L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.7 11 29 F,V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37Hallmark residue: F⁽¹⁾, H, I, L, Y 1.1 6 or V, preferably F⁽¹⁾ or Y 38 RR 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, TP, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, 1.3 5 R, S, L; preferably G⁽²⁾,E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾. 45 Hallmark residue: L⁽²⁾,R⁽³⁾, C, I, L, P, Q, V; 0.6 4 preferably L⁽²⁾ or R⁽³⁾ 46 E, V E, D, K,Q, V 0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, 1.9 9 M, R,S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A,G A, S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0.1 1 67 F F,L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.54 73 N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G,N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R,S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F,G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 QQ, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2  82a N, G N, D, G, H, S, T0.8 4  82b S S, N, D, G, R, T 1 6  82c L L, P, V 0.1 2 83 Hallmarkresidue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; 0.9 7 preferably K or R;most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V; 0.7 6preferably P 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 388 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 191 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H,K, N, R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 0.4 2 104 Hallmark residue: Gor D; preferably G 0.1 1 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or R: preferably Q or L⁽⁷⁾0.4 3 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can be defined as an amino acid sequence with the (general)structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-3;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequencewith the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 (it being understood that V_(HH) sequences will contain one or    more Hallmark residues; and that partially humanized Nanobodies will    usually, and preferably, [still] contain one or more Hallmark    residues [although it is also within the scope of the invention to    provide—where suitable in accordance with the invention—partially    humanized Nanobodies in which all Hallmark residues, but not one or    more of the other amino acid residues, have been humanized]; and    that in fully humanized Nanobodies, where suitable in accordance    with the invention, all amino acid residues at the positions of the    Hallmark residues will be amino acid residues that occur in a human    V_(H)3 sequence. As will be clear to the skilled person based on the    disclosure herein that such V_(HH) sequences, such partially    humanized Nanobodies with at least one Hallmark residue, such    partially humanized Nanobodies without Hallmark residues and such    fully humanized Nanobodies all form aspects of this invention);    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the single variable domains of SEQ ID NO's: 1    to 22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded;    and in which:-   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) or may be humanizedNanobodies. When the above Nanobody sequences are V_(HH) sequences, theymay be suitably humanized, as further described herein. When theNanobodies are partially humanized Nanobodies, they may optionally befurther suitably humanized, again as described herein.

TABLE A-9 Representative single variable domains for Nanobodies of theKERE, GLEW and P, R, S 103 group. KERE sequence no. 1 SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ ID NO:2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ ID NO: 3AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ IDNO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ IDNO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ IDNO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ IDNO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ IDNO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ IDNO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ IDNO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ IDNO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ IDNO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTVSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ IDNO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ IDNO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ IDNO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3SEQ ID NO: 22EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS The CDR's are indicated withXXXX

In particular, a Nanobody of the invention of the KERE group can be anamino acid sequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-10 Representative FW1 sequences for Nanobodies of theKERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO: 29QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 30EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE FW1 sequence no. 9 SEQ ID NO: 31AQELEESGGGLVQAGGSLRLSCAASGRTFNand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-11 Representative FW2 sequences for Nanobodies of theKERE-group. KERE FW2 sequence SEQ ID NO: 41 WFRQAPGKEREFVA no. 1 KEREFW2 sequence SEQ ID NO: 42 WFRQTPGREREFVA no. 2 KERE FW2 sequence SEQ IDNO: 43 WYRQAPGKQREMVA no. 3 KERE FW2 sequence SEQ ID NO: 44WYRQGPGKQRELVA no. 4 KERE FW2 sequence SEQ ID NO: 45 WIRQAPGKEREGVS no.5 KERE FW2 sequence SEQ ID NO: 46 WFREAPGKEREGIS no. 6 KERE FW2 sequenceSEQ ID NO: 47 WYRQAPGKERDLVA no. 7 KERE FW2 sequence SEQ ID NO: 48WFRQAPGKQREEVS no. 8 KERE FW2 sequence SEQ ID NO: 49 WFRQPPGKVREFVG no.9and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-12 Representative FW3 sequences for Nanobodies of theKERE-group. KERE FW3 sequence no. 1 SEQ ID NO: 50RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYF KERE FW3 sequence no. 2 SEQ ID NO: 51RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA KERE FW3 sequence no. 3 SEQ ID NO: 52RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA KERE FW3 sequence no. 4 SEQ ID NO: 53RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA KERE FW3 sequence no. 5 SEQ ID NO: 54RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAA KERE FW3 sequence no. 6 SEQ ID NO: 55RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA KERE FW3 sequence no. 7 SEQ ID NO: 56RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT KERE FW3 sequence no. 8 SEQ ID NO: 57RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAA KERE FW3 sequence no. 9 SEQ ID NO: 58RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA KERE FW3 sequence no. 10 SEQ ID NO: 59RFTISRDYAGNTAYLQMNSLKPEDTGVYYCATand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-13 Representative FW4 sequences for Nanobodies of theKERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS KERE FW4sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE FW4 sequence no. 3 SEQ IDNO: 62 RGQGTRVTVSS KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISSand in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are VHHsequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be VHH sequences or may behumanized Nanobodies. When the above Nanobody sequences are VHHsequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that,when an amino acid sequence as outlined above is generated by expressionof a nucleotide sequence, the first four single variable domains (i.e.amino acid residues 1-4 according to the Kabat numbering) may often bedetermined by the primer(s) that have been used to generate said nucleicacid. Thus, for determining the degree of amino acid identity, the firstfour amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 VHH sequences that the positions 27 to 30have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables A-5 to A-8) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. KERE FW1 sequence no. 10 SEQ ID NO: 32VESGGGLVQPGGSLRLSCAASG KERE FW1 sequence no. 11 SEQ ID NO: 33VDSGGGLVQAGDSLKLSCALTG KERE FW1 sequence no. 12 SEQ ID NO: 34VDSGGGLVQAGDSLRLSCAASG KERE FW1 sequence no. 13 SEQ ID NO: 35VDSGGGLVEAGGSLRLSCQVSE KERE FW1 sequence no. 14 SEQ ID NO: 36QDSGGGSVQAGGSLKLSCAASG KERE FW1 sequence no. 15 SEQ ID NO: 37VQSGGRLVQAGDSLRLSCAASE KERE FW1 sequence no. 16 SEQ ID NO: 38VESGGTLVQSGDSLKLSCASST KERE FW1 sequence no. 17 SEQ ID NO: 39MESGGDSVQSGGSLTLSCVASG KERE FW1 sequence no. 18 SEQ ID NO: 40QASGGGLVQAGGSLRLSCSASVand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the KERE-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be VHH sequences or may behumanized Nanobodies. When the above Nanobody sequences are VHHsequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-15 Representative FW1 sequences for Nanobodies of theGLEW-group. GLEW FW1 sequence no. 1 SEQ ID NO: 64QVQLVESGGGLVQPGGSLRLSCAASGFTFS GLEW FW1 sequence no. 2 SEQ ID NO: 65EVHLVESGGGLVRPGGSLRLSCAAFGFIFK GLEW FW1 sequence no. 3 SEQ ID NO: 66QVKLEESGGGLAQPGGSLRLSCVASGFTFS GLEW FW1 sequence no. 4 SEQ ID NO: 67EVQLVESGGGLVQPGGSLRLSCVCVSSGCT GLEW FW1 sequence no. 5 SEQ ID NO: 68EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-16 Representative FW2 sequences for Nanobodies of theGLEW-group. GLEW FW2 sequence no. SEQ ID NO: 72 WVRQAPGKVLEWVS 1 GLEWFW2 sequence no. SEQ ID NO: 73 WVRRPPGKGLEWVS 2 GLEW FW2 sequence no.SEQ ID NO: 74 WVRQAPGMGLEWVS 3 GLEW FW2 sequence no. SEQ ID NO: 75WVRQAPGKEPEWVS 4 GLEW FW2 sequence no. SEQ ID NO: 76 WVRQAPGKDQEWVS 5GLEW FW2 sequence no. SEQ ID NO: 77 WVRQAPGKAEEWVS 6 GLEW FW2 sequenceno. SEQ ID NO: 78 WVRQAPGKGLEWVA 7 GLEW FW2 sequence no. SEQ ID NO: 79WVRQAPGRATEWVS 8and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-17 Representative FW3 sequences for Nanobodies of theGLEW-group. GLEW FW3 sequence no. 1 SEQ ID NO: 80RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK GLEW FW3 sequence no. 2 SEQ ID NO: 81RFTISRDNARNTLYLQMDSLIPEDTALYYCAR GLEW FW3 sequence no. 3 SEQ ID NO: 82RFTSSRDNAKSTLYLQMNDLKPEDTALYYCAR GLEW FW3 sequence no. 4 SEQ ID NO: 83RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQR GLEW FW3 sequence no. 5 SEQ ID NO: 84RFTASRDNAKNTLYLQMNSLKSEDTARYYCAR GLEW FW3 sequence no. 6 SEQ ID NO: 85RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGRand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-18 Representative FW4 sequences for Nanobodies of theGLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS GLEW FW4sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW FW4 sequence no. 3 SEQ IDNO: 88 RGQGTLVTVSS GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSSGLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW FW4 sequence no.6 SEQ ID NO: 91 WGQGTQVTVSSand in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are VHHsequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. GLEW FW1 SEQ ID NO: 69VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW FW1 SEQ ID NO: 70EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1 SEQ ID NO: 71VESGGGLALPGGSLTLSCVFSG sequence no. 8and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the GLEW-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be VHH sequences or may behumanized Nanobodies. When the above Nanobody sequences are VHHsequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein. Inthe above Nanobodies, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are VHH sequencesor partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence thatis comprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-20 Representative FW1 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW1 sequence no. 1 SEQ ID NO: 92AVQLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1 sequence no. 2 SEQ ID NO:93 QVQLQESGGGMVQPGGSLRLSCAASGFDFG P, R, S 103 FW1 sequence no. 3 SEQ IDNO: 94 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK P, R, S 103 FW1 sequence no. 4 SEQID NO: 95 QVQLAESGGGLVQPGGSLKLSCAASRTIVS P, R, S 103 FW1 sequence no. 5SEQ ID NO: 96 QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequenceno. 6 SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRLSCVASGFTFS P, R, S 103 FW1sequence no. 7 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S 103FW1 sequence no. 8 SEQ ID NO: 99 EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-21 Representative FW2 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW2 sequence no. 1 SEQ ID NO: 102 WFRQAPGKEREFVAP, R, S 103 FW2 sequence no. 2 SEQ ID NO: 103 WVRQAPGKVLEWVS P, R, S 103FW2 sequence no. 3 SEQ ID NO: 104 WVRRPPGKGLEWVS P, R, S 103 FW2sequence no. 4 SEQ ID NO: 105 WIRQAPGKEREGVS P, R, S 103 FW2 sequenceno. 5 SEQ ID NO: 106 WVRQYPGKEPEWVS P, R, S 103 FW2 sequence no. 6 SEQID NO: 107 WFRQPPGKEHEFVA P, R, S 103 FW2 sequence no. 7 SEQ ID NO: 108WYRQAPGKRTELVA P, R, S 103 FW2 sequence no. 8 SEQ ID NO: 109WLRQAPGQGLEWVS P, R, S 103 FW2 sequence no. 9 SEQ ID NO: 110WLRQTPGKGLEWVG P, R, S 103 FW2 sequence no. 10 SEQ ID NO: 111WVRQAPGKAEEFVSand in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW3 sequence no. 1 SEQ ID NO: 112RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA P, R, S 103 FW3 sequence no. 2 SEQ IDNO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR P, R, S 103 FW3 sequence no. 3SEQ ID NO: 114 RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA P, R, S 103 FW3 sequenceno. 4 SEQ ID NO: 115 RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA P, R, S 103 FW3sequence no. 5 SEQ ID NO: 116 RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR P, R, S103 FW3 sequence no. 6 SEQ ID NO: 117 RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNLP, R, S 103 FW3 sequence no. 7 SEQ ID NO: 118RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR P, R, S 103 FW3 sequence no. 8 SEQ IDNO: 119 RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVand in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-23 Representative FW4 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW4 SEQ ID NO: 120 RGQGTQVTVSS sequence no. 1 P,R, S 103 FW4 SEQ ID NO: 121 LRGGTQVTVSS sequence no. 2 P, R, S 103 FW4SEQ ID NO: 122 GNKGTLVTVSS sequence no. 3 P, R, S 103 FW4 SEQ ID NO: 123SSPGTQVTVSS sequence no. 4 P, R, S 103 FW4 SEQ ID NO: 124 SSQGTLVTVSSsequence no. 5 P, R, S 103 FW4 SEQ ID NO: 125 RSRGIQVTVSS sequence no. 6and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are VHHsequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P,R,S 103 class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 SEQ ID NO:VESGGGLVQAGGSLRLSCAASG sequence no. 9 100 P, R, S 103 FW1 SEQ ID NO:AESGGGLVQPGGSLKLSCAASR sequence no. 10 101and in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of    Nanobodies of the P,R,S 103 class;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be VHH sequences or may behumanized Nanobodies. When the above Nanobody sequences are VHHsequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are VHHsequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the single variable domains of SEQ IDNO's: [255-269]. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said Nanobody and one or more of the sequencesof SEQ ID NO's: [255-269], in which the amino acid residues that formthe framework regions are disregarded. Such Nanobodies can be as furtherdescribed herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a Nanobody with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: [255-269] or fromthe group consisting of from single variable domains that have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with at least one ofthe single variable domains of SEQ ID NO's: [255-269].

Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing    substitution as defined herein) is preferably, and compared to the    corresponding amino acid sequence of SEQ ID NO's: [255-269], a    conservative amino acid substitution, (as defined herein);    and/or:-   ii) its amino acid sequence preferably contains either only amino    acid substitutions, or otherwise preferably no more than 5,    preferably no more than 3, and more preferably only 1 or 2 amino    acid deletions or insertions, compared to the corresponding amino    acid sequence of SEQ ID NO's: [255-269];    and/or-   iii) the CDR's may be CDR's that are derived by means of affinity    maturation, for example starting from the CDR's of to the    corresponding amino acid sequence of SEQ ID NO's: [255-269].

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobodies of the invention (and singlevariable domains of the invention comprising the same):

-   -   bind to members of the Notch signalling pathway with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and        preferably 10⁷ to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to members of the Notch signalling pathway with a        k_(on)-rate of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,        preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably        between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹        and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to members of the Notch signalling pathway with a k_(off)        rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a        near irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies ofthe invention are such that the Nanobodies of the invention will bind tomembers of the Notch signalling pathway with an affinity less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at least one of the Hallmark residuesin FR2 and/or FR4 (again, including those at positions 108, 103 and/or45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's [255-269]. Thus,according to one aspect of the invention, the term “Nanobody of theinvention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables A-5 toA-8 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5 to A-8 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to members of theNotch signalling pathway with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs: [255-269].

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “human-like”, while still retaining the favorableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P,R,S-103group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein). This can generally be performed usingmethods and techniques known per se, which will be clear to the skilledperson, for example from the handbooks and references cited herein, thebackground art cited herein and/or from the further description herein.Alternatively, a nucleic acid encoding the desired analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can then be expressed as described herein. Yetanother technique may involve combining one or more naturally occurringand/or synthetic nucleic acid sequences each encoding a part of thedesired analog, and then expressing the combined nucleic acid sequenceas described herein. Also, the analogs can be provided using chemicalsynthesis of the pertinent amino acid sequence using techniques forpeptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNanobodies of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i.e. singlevariable domains or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables A-5-A-8. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal V_(H) domain). Generally, however, such camelizingsubstitutions are preferably such that the resulting an amino acidsequence at least contains (a) a Q at position 108; and/or (b) a chargedamino acid or a cysteine residue at position 45 and preferably also an Eat position 44, and more preferably E at position 44 and R at position45; and/or (c) P, R or S at position 103; and optionally one or morefurther camelizing substitutions. More preferably, the camelizingsubstitutions are such that they result in a Nanobody of the inventionand/or in an analog thereof (as defined herein), such as in a humanizedanalog and/or preferably in an analog that is as defined in thepreceding paragraphs.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NO's: [255-269]. Thus, according to one aspect of theinvention, the term “Nanobody of the invention” in its broadest sensealso covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have single variable domains in which,compared to the amino acid sequence of the corresponding full lengthNanobody of the invention (or analog thereof), one or more of the aminoacid residues at the N-terminal end, one or more amino acid residues atthe C-terminal end, one or more contiguous internal amino acid residues,or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to membersof the Notch signalling pathway with an affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein for the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length Nanobody of the invention, i.e. asfor example described in the International application WO 03/050531(Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs[255-269].

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/or singlevariable domains of the invention; or any combination of two or more ofthe foregoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin-avidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and single variable domains of the inventionmay for example be used for in vitro, in vivo or in situ assays(including immunoassays known per se such as ELISA, RIA, EIA and other“sandwich assays”, etc.) as well as in vivo diagnostic and imagingpurposes, depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethylenetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to members of theNotch signalling pathway with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins or singlevariable domains that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such single variable        domains will be clear to the skilled person. Some non-limiting        examples are the small peptide vectors (“Pep-trans vectors”)        described in WO 03/026700 and in Temsamani et al., Expert Opin.        Biol. Ther., 1, 773 (2001); Temsamani and Vidal, Drug Discov.        Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther.,        296, 124-131 (2001), and the membrane translocator sequence        described by Zhao et al., Apoptosis, 8, 631-637 (2003).        C-terminal and N-terminal single variable domains for        intracellular targeting of antibody fragments are for example        described by Cardinale et al., Methods, 34, 171 (2004). Other        suitable techniques for intracellular targeting involve the        expression and/or use of so-called “intrabodies” comprising a        Nanobody of the invention, as mentioned below;    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the Nanobody, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        Nanobody sequence (for this purpose, the tag may optionally be        linked to the Nanobody sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutathione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.

According to another aspect, a polypeptide of the invention comprises aNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said Nanobody of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further single variable domains may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the Nanobody of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such single variable domains will be clear to the skilledperson, and may generally comprise all single variable domains that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the single variable domains of theinvention, compared to the Nanobody of the invention per se. Somenon-limiting examples of such single variable domains are serumproteins, such as human serum albumin (see for example WO 00/27435) orhaptenic molecules (for example haptens that are recognized bycirculating antibodies, see for example WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to the U.S. provisional application 60/788,256 ofAblynx N.V. entitled “Albumin derived amino acid sequence, use thereoffor increasing the half-life of therapeutic proteins and of othertherapeutic proteins and entities, and constructs comprising the same”filed on Mar. 31, 2006 (see also PCT/EP2007/002817).

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such singlevariable domains for example include the Nanobodies described below, aswell as the small peptides and binding proteins described in WO91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO03/002609 and WO 04/003019. Reference is also made to Harmsen et al.,Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well asto the following the U.S. provisional applications 60/843,349 (see alsoPCT/EP2007/059475), 60/850,774 (see also PCT/EP2007/060849), 60/850,775(see also PCT/EP2007/060850) by Ablynx N.V. mentioned herein and USprovisional application of Ablynx N.V. entitled “Peptides capable ofbinding to serum proteins” filed on Dec. 5, 2006 ((see alsoPCT/EP2007/063348).

Such single variable domains may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such single variabledomains may be single variable domains that are directed against (human)serum albumin and single variable domains that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or singlevariable domains that are capable of binding to amino acid residues onserum albumin that do not form part of domain III of serum albumin (seeagain for example WO 06/0122787); single variable domains that have orcan provide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V. entitled “Serum albumin bindingproteins with long half-lives” filed on Sep. 8, 2006; see alsoPCT/EP2007/059475); single variable domains against human serum albuminthat are cross-reactive with serum albumin from at least one species ofmammal, and in particular with at least one species of primate (such as,without limitation, monkeys from the genus Macaca (such as, and inparticular, cynomolgus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus), reference is againmade to the U.S. provisional application 60/843,349 andPCT/EP2007/059475); single variable domains that can bind to serumalbumin in a pH independent manner (see for example the U.S. provisionalapplication 60/850,774 by Ablynx N.V. entitled “Single variable domainsthat bind to serum proteins in a manner that is essentially independentof the pH, compounds comprising the same, and uses thereof”, filed onOct. 11, 2006; see also and PCT/EP2007/059475) and/or single variabledomains that are conditional binders (see for example the U.S.provisional application 60/850,775 by Ablynx N.V. entitled “Singlevariable domains that bind to a desired molecule in a conditionalmanner”, filed on Oct. 11, 2006; see also PCT/EP2007/060850).

According to another aspect, the one or more further single variabledomains may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a Nanobody linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody of the invention. Also, two Nanobodies couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more Nanobodies of the invention may be linked (optionally via asuitable linker or hinge region) to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors. For example, for this purpose, and withoutbeing limited thereto, the one or more further single variable domainsmay comprise one or more C_(H)2 and/or C_(H)3 domains of an antibody,such as from a heavy chain antibody (as described herein) and morepreferably from a conventional human 4-chain antibody; and/or may form(part of) and Fc region, for example from IgG (e.g. from IgG1, IgG2,IgG3 or IgG4), from IgE or from another human Ig such as IgA, IgD orIgM. For example, WO 94/04678 describes heavy chain antibodiescomprising a Camelid V_(HH) domain or a humanized derivative thereof(i.e. a Nanobody), in which the Camelidae C_(H)2 and/or C_(H)3 domainhave been replaced by human C_(H)2 and C_(H)3 domains, so as to providean immunoglobulin that consists of 2 heavy chains each comprising aNanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain),which immunoglobulin has the effector function provided by the C_(H)2and C_(H)3 domains and which immunoglobulin can function without thepresence of any light chains. Other single variable domains that can besuitably linked to the Nanobodies of the invention so as to provide aneffector function will be clear to the skilled person, and may be chosenon the basis of the desired effector function(s). Reference is forexample made to WO 04/058820, WO 99/42077, WO 02/056910 and WO05/017148, as well as the review by Holliger and Hudson, supra; and tothe non-prepublished US provisional application by Ablynx N.V. entitled“Constructs comprising single variable domains and an Fc portion derivedfrom IgE” which has a filing date of Dec. 4, 2007. Coupling of aNanobody of the invention to an Fc portion may also lead to an increasedhalf-life, compared to the corresponding Nanobody of the invention. Forsome applications, the use of an Fc portion and/or of constant domains(i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-lifewithout any biologically significant effector function may also besuitable or even preferred. Other suitable constructs comprising one ormore Nanobodies and one or more constant domains with increasedhalf-life in vivo will be clear to the skilled person, and may forexample comprise two Nanobodies linked to a C_(H)3 domain, optionallyvia a linker sequence. Generally, any fusion protein or derivatives withincreased half-life will preferably have a molecular weight of more than50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more single variable domains of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al., J Biol Chem 1996 2717494, describe monomeric Fcε chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use). Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Bivalent/multivalent, bispecific/multispecific orbiparatopic/multiparatopic single variable domains of the invention mayalso be linked to Fc portions, in order to provide polypeptideconstructs of the type that is described in the non-prepublished USprovisional application entitled “immunoglobulin constructs” filed onDec. 4, 2007.

The further single variable domains may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such single variable domainswill be clear to the skilled person, and for example include, but arenot limited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the antibody fragments known per sethat can be used to express or produce the Nanobodies and singlevariable domains of the invention as so-called “intrabodies”, forexample as described in WO 94/02610, WO 95/22618, U.S. Pat. No.7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andsingle variable domains which can be linked to a Nanobody of theinvention to provide—for example—a cytotoxic polypeptide of theinvention will be clear to the skilled person and can for example befound in the prior art cited above and/or in the further descriptionherein. One example is the so-called ADEPT™ technology described in WO03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther single variable domains comprise at least one further Nanobody,so as to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein). Single variable domains of the invention that comprisetwo or more Nanobodies, of which at least one is a Nanobody of theinvention, will also be referred to herein as “multivalent” singlevariable domains of the invention, and the Nanobodies present in suchsingle variable domains will also be referred to herein as being in a“multivalent format”. For example a “bivalent” polypeptide of theinvention comprises two Nanobodies, optionally linked via a linkersequence, whereas a “trivalent” polypeptide of the invention comprisesthree Nanobodies, optionally linked via two linker sequences; etc.; inwhich at least one of the Nanobodies present in the polypeptide, and upto all of the Nanobodies present in the polypeptide, is/are a Nanobodyof the invention.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different, and may be directed against thesame antigen or antigenic determinant (for example against the samepart(s) or epitope(s) or against different parts or epitopes) or mayalternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide of the invention may comprise (a) two identicalNanobodies; (b) a first Nanobody directed against a first antigenicdeterminant of a protein or antigen and a second Nanobody directedagainst the same antigenic determinant of said protein or antigen whichis different from the first Nanobody; (c) a first Nanobody directedagainst a first antigenic determinant of a protein or antigen and asecond Nanobody directed against another antigenic determinant of saidprotein or antigen; or (d) a first Nanobody directed against a firstprotein or antigen and a second Nanobody directed against a secondprotein or antigen (i.e. different from said first antigen). Similarly,a trivalent polypeptide of the invention may, for example and withoutbeing limited thereto. comprise (a) three identical Nanobodies; (b) twoidentical Nanobody against a first antigenic determinant of an antigenand a third Nanobody directed against a different antigenic determinantof the same antigen; (c) two identical Nanobody against a firstantigenic determinant of an antigen and a third Nanobody directedagainst a second antigen different from said first antigen; (d) a firstNanobody directed against a first antigenic determinant of a firstantigen, a second Nanobody directed against a second antigenicdeterminant of said first antigen and a third Nanobody directed againsta second antigen different from said first antigen; or (e) a firstNanobody directed against a first antigen, a second Nanobody directedagainst a second antigen different from said first antigen, and a thirdNanobody directed against a third antigen different from said first andsecond antigen.

Single variable domains of the invention that contain at least twoNanobodies, in which at least one Nanobody is directed against a firstantigen (i.e. against members for the Notch signalling pathway,) and atleast one Nanobody is directed against a second antigen (i.e. differentfrom members for the Notch signalling pathway,), will also be referredto as “multispecific” single variable domains of the invention, and theNanobodies present in such single variable domains will also be referredto herein as being in a “multispecific format”. Thus, for example, a“bispecific” polypeptide of the invention is a polypeptide thatcomprises at least one Nanobody directed against a first antigen (i.e.members for the Notch signalling pathway,) and at least one furtherNanobody directed against a second antigen (i.e. different from membersfor the Notch signalling pathway,), whereas a “trispecific” polypeptideof the invention is a polypeptide that comprises at least one Nanobodydirected against a first antigen (i.e. members for the Notch signallingpathway,), at least one further Nanobody directed against a secondantigen (i.e. different from members for the Notch signalling pathway,)and at least one further Nanobody directed against a third antigen (i.e.different from both members for the Notch signalling pathway, and thesecond antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against members for theNotch signalling pathway, and a second Nanobody directed against asecond antigen, in which said first and second Nanobody may optionallybe linked via a linker sequence (as defined herein); whereas atrispecific polypeptide of the invention in its simplest form is atrivalent polypeptide of the invention (as defined herein), comprising afirst Nanobody directed against members for the Notch signallingpathway, a second Nanobody directed against a second antigen and a thirdNanobody directed against a third antigen, in which said first, secondand third Nanobody may optionally be linked via one or more, and inparticular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againstmembers for the Notch signalling pathway, and any number of Nanobodiesdirected against one or more antigens different from members for theNotch signalling pathway.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the single variable domains of the invention may have someinfluence on the properties of the final polypeptide of the invention(including but not limited to the affinity, specificity or avidity formembers for the Notch signalling pathway, or against the one or moreother antigens), said order or arrangement is usually not critical andmay be suitably chosen by the skilled person, optionally after somelimited routine experiments based on the disclosure herein. Thus, whenreference is made to a specific multivalent or multispecific polypeptideof the invention, it should be noted that this encompasses any order orarrangements of the relevant Nanobodies, unless explicitly indicatedotherwise.

Finally, it is also within the scope of the invention that the singlevariable domains of the invention contain two or more Nanobodies and oneor more further single variable domains (as mentioned herein).

For multivalent and multispecific single variable domains containing oneor more V_(HH) domains and their preparation, reference is also made toConrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001;Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; aswell as to for example WO 96/34103 and WO 99/23221. Some other examplesof some specific multispecific and/or multivalent polypeptide of theinvention can be found in the applications by Ablynx N.V. referred toherein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. SuchNanobodies may for example be Nanobodies that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, Nanobodies that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example Nanobody VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, Nanobodies against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,Nanobodies against human serum albumin or human IgG will usually bepreferred). Nanobodies that provide for increased half-life and that canbe used in the single variable domains of the invention include theNanobodies directed against serum albumin that are described in WO04/041865, in WO 06/122787 and in the further patent applications byAblynx N.V., such as those mentioned above.

For example, the some preferred Nanobodies that provide for increasedhalf-life for use in the present invention include Nanobodies that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see for example WO06/0122787); Nanobodies that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); Nanobodies that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V mentioned herein; see alsoPCT/EP2007/059475); Nanobodies against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) (see for example the U.S.provisional application 60/843,349 by Ablynx N.V; see alsoPCT/EP2007/059475)); Nanobodies that can bind to serum albumin in a pHindependent manner (see for example the U.S. provisional application60/850,774 by Ablynx N.V. mentioned herein) and/or Nanobodies that areconditional binders (see for example the U.S. provisional application60/850,775 by Ablynx N.V.; see also PCT/EP2007/060850).

Some particularly preferred Nanobodies that provide for increasedhalf-life and that can be used in the single variable domains of theinvention include the Nanobodies ALB-1 to ALB-10 disclosed in WO06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO06/122787) is particularly preferred.

According to a specific, but non-limiting aspect of the invention, thesingle variable domains of the invention contain, besides the one ormore Nanobodies of the invention, at least one Nanobody against humanserum albumin.

Generally, any single variable domains of the invention with increasedhalf-life that contain one or more Nanobodies of the invention, and anyderivatives of Nanobodies of the invention or of such single variabledomains that have an increased half-life, preferably have a half-lifethat is at least 1.5 times, preferably at least 2 times, such as atleast 5 times, for example at least 10 times or more than 20 times,greater than the half-life of the corresponding Nanobody of theinvention per se. For example, such a derivative or single variabledomains with increased half-life may have a half-life that is increasedwith more than 1 hours, preferably more than 2 hours, more preferablymore than 6 hours, such as more than 12 hours, or even more than 24, 48or 72 hours, compared to the corresponding Nanobody of the invention perse.

In a preferred, but non-limiting aspect of the invention, suchderivatives or single variable domains may exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, such derivatives or single variable domains may havea half-life of at least 5 days (such as about 5 to 10 days), preferablyat least 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

According to one aspect of the invention the single variable domains arecapable of binding to one or more molecules which can increase thehalf-life of the polypeptide in vivo.

The single variable domains of the invention are stabilised in vivo andtheir half-life increased by binding to molecules which resistdegradation and/or clearance or sequestration. Typically, such moleculesare naturally occurring proteins which themselves have a long half-lifein vivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one Nanobody of theinvention and at least one Nanobody that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the Nanobody to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such Nanobodies include Nanobodies thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In the single variable domains of the invention, the one or moreNanobodies and the one or more single variable domains may be directlylinked to each other (as for example described in WO 99/23221) and/ormay be linked to each other via one or more suitable spacers or linkers,or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificsingle variable domains will be clear to the skilled person, and maygenerally be any linker or spacer used in the art to link amino acidsequences. Preferably, said linker or spacer is suitable for use inconstructing proteins or single variable domains that are intended forpharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular single variable domains of between 1 and 50, preferablybetween 1 and 30, such as between 1 and 10 amino acid residues. Somepreferred examples of such single variable domains include gly-serlinkers, for example of the type (gly_(x)ser_(y))_(z), such as (forexample (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and theGS30, GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for members for the Notch signalling pathway, orfor one or more of the other antigens. Based on the disclosure herein,the skilled person will be able to determine the optimal linker(s) foruse in a specific polypeptide of the invention, optionally after somelimited routine experiments.

For example, in multivalent single variable domains of the inventionthat comprise Nanobodies directed against a multimeric antigen (such asa multimeric receptor or other protein), the length and flexibility ofthe linker are preferably such that it allows each Nanobody of theinvention present in the polypeptide to bind to the antigenicdeterminant on each of the subunits of the multimer. Similarly, in amultispecific polypeptide of the invention that comprises Nanobodiesdirected against two or more different antigenic determinants on thesame antigen (for example against different epitopes of an antigenand/or against different subunits of a multimeric receptor, channel orprotein), the length and flexibility of the linker are preferably suchthat it allows each Nanobody to bind to its intended antigenicdeterminant. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thesingle variable domains of the invention, and/or provide one or moresites for the formation of derivatives and/or for the attachment offunctional groups (e.g. as described herein for the derivatives of theNanobodies of the invention). For example, linkers containing one ormore charged amino acid residues (see Table A-2 above) can provideimproved hydrophilic properties, whereas linkers that form or containsmall epitopes or tags can be used for the purposes of detection,identification and/or purification. Again, based on the disclosureherein, the skilled person will be able to determine the optimal linkersfor use in a specific polypeptide of the invention, optionally aftersome limited routine experiments.

Finally, when two or more linkers are used in the single variabledomains of the invention, these linkers may be the same or different.Again, based on the disclosure herein, the skilled person will be ableto determine the optimal linkers for use in a specific polypeptide ofthe invention, optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the single variable domainsof the invention, which may be essentially analogous to the derivativesof the Nanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or single variable domains that“essentially consist” of a polypeptide of the invention (in which thewording “essentially consist of” has essentially the same meaning asindicated hereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, single variable domains andnucleic acids of the invention can be prepared in a manner known per se,as will be clear to the skilled person from the further descriptionherein. For example, the Nanobodies and polypeptides of the inventioncan be prepared in any manner known per se for the preparation ofantibodies and in particular for the preparation of antibody fragments(including but not limited to (single) domain antibodies and ScFvfragments). Some preferred, but non-limiting methods for preparing theamino acid sequences, Nanobodies, single variable domains and nucleicacids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, Nanobody or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, Nanobody and/or    polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the single variabledomains for the single variable domains of the invention given herein,and/or can be isolated from a suitable natural source. To provideanalogs, nucleotide sequences encoding naturally occurring V_(HH)domains can for example be subjected to site-directed mutagenesis, so atto provide a nucleic acid of the invention encoding said analog. Also,as will be clear to the skilled person, to prepare a nucleic acid of theinvention, also several nucleotide sequences, such as at least onenucleotide sequence encoding a Nanobody and for example nucleic acidsencoding one or more linkers can be linked together in a suitablemanner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of members of the Notch signalling pathway as atemplate. These and other techniques will be clear to the skilledperson, and reference is again made to the standard handbooks, such asSambrook et al. and Ausubel et al., mentioned above, as well as theExamples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described herein); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used. For example, regulatory sequences, promoters and terminators    known per se for the expression and production of antibodies and    antibody fragments (including but not limited to (single) domain    antibodies and ScFv fragments) may be used in an essentially    analogous manner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promoter). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al., (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The amino acid sequences, Nanobodies and single variable domains of theinvention can also be introduced and expressed in one or more cells,tissues or organs of a multicellular organism, for example forprophylactic and/or therapeutic purposes (e.g. as a gene therapy). Forthis purpose, the nucleotide sequences of the invention may beintroduced into the cells or tissues in any suitable way, for example assuch (e.g. using liposomes) or after they have been inserted into asuitable gene therapy vector (for example derived from retroviruses suchas adenovirus, or parvoviruses such as adeno-associated virus). As willalso be clear to the skilled person, such gene therapy may be performedin vivo and/or in situ in the body of a patient by administering anucleic acid of the invention or a suitable gene therapy vector encodingthe same to the patient or to specific cells or a specific tissue ororgan of the patient; or suitable cells (often taken from the body ofthe patient to be treated, such as explanted lymphocytes, bone marrowaspirates or tissue biopsies) may be treated in vitro with a nucleotidesequence of the invention and then be suitably (re-)introduced into thebody of the patient. All this can be performed using gene therapyvectors, techniques and delivery systems which are well known to theskilled person, and for example described in Culver, K. W., “GeneTherapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York,N.Y.); Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ.Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma,Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser,Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36;Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811(1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang,Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat.No. 5,580,859; US 5,5895466; or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640. For example, in situ expression of ScFvfragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and ofdiabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, Nanobodies and single variable domains of theinvention can for example also be produced in the milk of transgenicmammals, for example in the milk of rabbits, cows, goats or sheep (seefor example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S.Pat. No. 6,849,992 for general techniques for introducing transgenesinto mammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or tubers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and single variabledomains of the invention can also be expressed and/or produced incell-free expression systems, and suitable examples of such systems willbe clear to the skilled person. Some preferred, but non-limitingexamples include expression in the wheat germ system; in rabbitreticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the single variable domains based thereon can be prepared throughexpression in a suitable bacterial system, and suitable bacterialexpression systems, vectors, host cells, regulatory elements, etc., willbe clear to the skilled person, for example from the references citedabove. It should however be noted that the invention in its broadestsense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thesingle variable domains of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person, singlevariable domains of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, Nanobody or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, Nanobodies and the single variable domains of the invention,the amino acid sequences, Nanobodies and single variable domains of theinvention can be produced either intracellullarly (e.g. in the cytosol,in the periplasma or in inclusion bodies) and then isolated from thehost cells and optionally further purified; or can be producedextracellularly (e.g. in the medium in which the host cells arecultured) and then isolated from the culture medium and optionallyfurther purified. When eukaryotic host cells are used, extracellularproduction is usually preferred since this considerably facilitates thefurther isolation and downstream processing of the Nanobodies andproteins obtained. Bacterial cells such as the strains of E. colimentioned above normally do not secrete proteins extracellularly, exceptfor a few classes of proteins such as toxins and hemolysin, andsecretory production in E. coli refers to the translocation of proteinsacross the inner membrane to the periplasmic space. Periplasmicproduction provides several advantages over cytosolic production. Forexample, the N-terminal amino acid sequence of the secreted product canbe identical to the natural gene product after cleavage of the secretionsignal sequence by a specific signal peptidase. Also, there appears tobe much less protease activity in the periplasm than in the cytoplasm.In addition, protein purification is simpler due to fewer contaminatingproteins in the periplasm. Another advantage is that correct disulfidebonds may form because the periplasm provides a more oxidativeenvironment than the cytoplasm. Proteins overexpressed in E. coli areoften found in insoluble aggregates, so-called inclusion bodies. Theseinclusion bodies may be located in the cytosol or in the periplasm; therecovery of biologically active proteins from these inclusion bodiesrequires a denaturation/refolding process. Many recombinant proteins,including therapeutic proteins, are recovered from inclusion bodies.Alternatively, as will be clear to the skilled person, recombinantstrains of bacteria that have been genetically modified so as to secretea desired protein, and in particular an amino acid sequence, Nanobody ora polypeptide of the invention, can be used.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been produced intracellularlyand that has been isolated from the host cell, and in particular from abacterial cell or from an inclusion body in a bacterial cell. Accordingto another non-limiting aspect of the invention, the amino acidsequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left—(PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7 promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I);    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);

-   vectors for expression in yeast or other fungal cells: pYES2    (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Suc), etc.,    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig η-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the single variable domains of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, thesingle variable domains of the invention may be expressed in aconstitutive manner, in a transient manner, or only when suitablyinduced.

It will also be clear to the skilled person that the amino acidsequence, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the single variable domains of theinvention may be formulated as a pharmaceutical preparation orcompositions comprising at least one polypeptide of the invention and atleast one pharmaceutically acceptable carrier, diluent or excipientand/or adjuvant, and optionally one or more further pharmaceuticallyactive single variable domains and/or compounds. By means ofnon-limiting examples, such a formulation may be in a form suitable fororal administration, for parenteral administration (such as byintravenous, intramuscular or subcutaneous injection or intravenousinfusion), for topical administration, for administration by inhalation,by a skin patch, by an implant, by a suppository, etc. Such suitableadministration forms—which may be solid, semi-solid or liquid, dependingon the manner of administration—as well as methods and carriers for usein the preparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, Nanobodies and single variabledomains of the invention can be formulated and administered in anysuitable manner known per se, for which reference is for example made tothe general background art cited above (and in particular to WO04/041862, WO 04/041863, WO 04/041865 and WO 04/041867) as well as tothe standard handbooks, such as Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990) or Remington, theScience and Practice of Pharmacy, 21st Edition, Lippincott Williams andWilkins (2005).

For example, the amino acid sequences, Nanobodies and single variabledomains of the invention may be formulated and administered in anymanner known per se for conventional antibodies and antibody fragments(including ScFv's and diabodies) and other pharmaceutically activeproteins. Such formulations and methods for preparing the same will beclear to the skilled person, and for example include preparationssuitable for parenteral administration (for example intravenous,intraperitoneal, subcutaneous, intramuscular, intraluminal,intra-arterial or intrathecal administration) or for topical (i.e.transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and single variable domains of theinvention can also be administered using gene therapy methods ofdelivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated byreference in its entirety. Using a gene therapy method of delivery,primary cells transfected with the gene encoding an amino acid sequence,Nanobody or polypeptide of the invention can additionally be transfectedwith tissue specific promoters to target specific organs, tissue,grafts, tumors, or cells and can additionally be transfected with signaland stabilization sequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and single variable domainsof the invention may be systemically administered, e.g., orally, incombination with a pharmaceutically acceptable vehicle such as an inertdiluent or an assimilable edible carrier. They may be enclosed in hardor soft shell gelatin capsules, may be compressed into tablets, or maybe incorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, Nanobodies andsingle variable domains of the invention may be combined with one ormore excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of the amino acid sequence, Nanobody or polypeptide of theinvention. Their percentage in the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, Nanobodies and single variable domainsof the invention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and single variable domains of the invention may beincorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and single variable domains of theinvention may also be administered intravenously or intraperitoneally byinfusion or injection. Solutions of the amino acid sequences, Nanobodiesand single variable domains of the invention or their salts can beprepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, Nanobodies and single variable domains of the inventionin the required amount in the appropriate solvent with various of theother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies andsingle variable domains of the invention may be applied in pure form,i.e., when they are liquids. However, it will generally be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, Nanobodies and singlevariable domains of the invention can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, Nanobodies and single variable domainsof the invention to the skin are known to the art; for example, seeJacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and singlevariable domains of the invention can be determined by comparing theirin vitro activity, and in vivo activity in animal models. Methods forthe extrapolation of effective dosages in mice, and other animals, tohumans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies andsingle variable domains of the invention in a liquid composition, suchas a lotion, will be from about 0.1-25 wt-%, preferably from about0.5-10 wt-%. The concentration in a semi-solid or solid composition suchas a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5wt-%.

The amount of the amino acid sequences, Nanobodies and single variabledomains of the invention required for use in treatment will vary notonly with the particular amino acid sequence, Nanobody or polypeptideselected but also with the route of administration, the nature of thecondition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. Also the dosage of the amino acid sequences, Nanobodies andsingle variable domains of the invention varies depending on the targetcell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one cancer, neurodegenerative diseases andimmunomodulatory diseases, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of an aminoacid sequence of the invention, of a Nanobody of the invention, of apolypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with members for theNotch signalling pathway, with its biological or pharmacologicalactivity, and/or with the biological pathways or signalling in whichmembers of the Notch signalling pathway is involved, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can be treatedby modulating members for the Notch signalling pathway, its biologicalor pharmacological activity, and/or the biological pathways orsignalling in which members of the Notch signalling pathway is involved,said method comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, said pharmaceutically effective amount may be an amountthat is sufficient to modulate members for the Notch signalling pathway,its biological or pharmacological activity, and/or the biologicalpathways or signalling in which members of the Notch signalling pathwayis involved; and/or an amount that provides a level of the amino acidsequence of the invention, of a Nanobody of the invention, of apolypeptide of the invention in the circulation that is sufficient tomodulate members for the Notch signalling pathway, its biological orpharmacological activity, and/or the biological pathways or signallingin which members of the Notch signalling pathway is involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,a Nanobody of the invention or a polypeptide of the invention to apatient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of a Nanobody of the invention, ofa polypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the above methods, the amino acid sequences, Nanobodies and/or singlevariable domains of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, Nanobodies and/or single variable domains of theinvention and/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, Nanobodies and/or single variable domains ofthe invention and/or the compositions comprising the same areadministered according to a regime of treatment that is suitable forpreventing and/or treating the disease or disorder to be prevented ortreated. The clinician will generally be able to determine a suitabletreatment regimen, depending on factors such as the disease or disorderto be prevented or treated, the severity of the disease to be treatedand/or the severity of the symptoms thereof, the specific amino acidsequence, Nanobody or polypeptide of the invention to be used, thespecific route of administration and pharmaceutical formulation orcomposition to be used, the age, gender, weight, diet, general conditionof the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies and/or single variable domainsof the invention, or of one or more compositions comprising the same, inone or more pharmaceutically effective amounts or doses. The specificamount(s) or doses to be administered can be determined by theclinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, Nanobodies and singlevariable domains of the invention will generally be administered in anamount between 1 gram and 0.01 microgram per kg body weight per day,preferably between 0.1 gram and 0.1 microgram per kg body weight perday, such as about 1, 10, 100 or 1000 microgram per kg body weight perday, either continuously (e.g. by infusion), as a single daily dose oras multiple divided doses during the day. The clinician will generallybe able to determine a suitable daily dose, depending on the factorsmentioned herein. It will also be clear that in specific cases, theclinician may choose to deviate from these amounts, for example on thebasis of the factors cited above and his expert judgment. Generally,some guidance on the amounts to be administered can be obtained from theamounts usually administered for comparable conventional antibodies orantibody fragments against the same target administered via essentiallythe same route, taking into account however differences inaffinity/avidity, efficacy, biodistribution, half-life and similarfactors well known to the skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences,Nanobodies and/or single variable domains of the invention incombination.

The Nanobodies, single variable domains of the invention may also beused in combination with one or more further pharmaceutically activecompounds or principles, i.e. as a combined treatment regimen, which mayor may not lead to a synergistic effect. Again, the clinician will beable to select such further compounds or principles, as well as asuitable combined treatment regimen, based on the factors cited aboveand his expert judgement.

In particular, the amino acid sequences, Nanobodies and single variabledomains of the invention may be used in combination with otherpharmaceutically active compounds or principles that are or can be usedfor the prevention and/or treatment of the diseases and disorders citedherein, as a result of which a synergistic effect may or may not beobtained. Examples of such compounds and principles, as well as routes,methods and pharmaceutical formulations or compositions foradministering them will be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone cancer, neurodegenerative diseases and immunomodulatory diseases;and/or for use in one or more of the methods of treatment mentionedherein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, Nanobody or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment ofcancer, neurodegenerative diseases and immunomodulatory diseases, and inparticular for the prevention and treatment of one or more of thediseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies or single variable domains of the invention mayalso be suitably combined with one or more other active principles, suchas those mentioned herein.

Finally, although the use of the Nanobodies of the invention (as definedherein) and of the single variable domains of the invention is muchpreferred, it will be clear that on the basis of the description herein,the skilled person will also be able to design and/or generate, in ananalogous manner, other in particular (single) domain antibodies againstmembers for the Notch signalling pathway, as well as single variabledomains comprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., ProteinScience (2004), 13:1882-1891; Ewert et al., Methods, 2004 October;34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7):773-783; O'Brien and Jones, Methods Mol. Biol. 2003:207:81-100; Skerra,J. Mol. Recognit. 2000:13:167-187, and Saerens et al., J. Mol. Biol.2005 Sep. 23; 352 (3):597-607, and the further references cited therein.For example, techniques known per se for grafting mouse or rat CDR'sonto human frameworks and scaffolds can be used in an analogous mannerto provide chimeric proteins comprising one or more of the CDR's of theNanobodies of the invention and one or more human framework regions orsequences.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example from Nanobodies (preferred), V_(H) domains fromconventional antibodies (and in particular from human antibodies), heavychain antibodies, conventional 4-chain antibodies (such as conventionalhuman 4-chain antibodies) or other immunoglobulin sequences directedagainst members for the Notch signalling pathway. Such immunoglobulinsequences directed against members of the Notch signalling pathway canbe generated in any manner known per se, as will be clear to the skilledperson, i.e. by immunization with members of the Notch signallingpathway or by screening a suitable library of immunoglobulin sequenceswith members for the Notch signalling pathway, or any suitablecombination thereof. Optionally, this may be followed by techniques suchas random or site-directed mutagenesis and/or other techniques foraffinity maturation known per se. Suitable techniques for generatingsuch immunoglobulin sequences will be clear to the skilled person, andfor example include the screening techniques reviewed by Hoogenboom,Nature Biotechnology, 23, 9, 1105-1116 (2005) Other techniques forgenerating immunoglobulins against a specified target include forexample the Nanoclone technology (as for example described in thepublished US patent application 2006-0211088), so-called SLAM technology(as for example described in the European patent application 0 542 810),the use of transgenic mice expressing human immunoglobulins or thewell-known hybridoma techniques (see for example Larrick et al,Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be usedto generate immunoglobulins against members for the Notch signallingpathway, and the CDR's of such immunoglobulins can be used in theNanobodies of the invention, i.e. as outlined above. For example, thesequence of such a CDR can be determined, synthesized and/or isolated,and inserted into the sequence of a Nanobody of the invention (e.g. soas to replace the corresponding native CDR), all using techniques knownper se such as those described herein, or Nanobodies of the inventioncontaining such CDR's (or nucleic acids encoding the same) can besynthesized de novo, again using the techniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, single variabledomains, nucleic acids, genetic constructs and hosts and host cells ofthe invention will be clear to the skilled person based on thedisclosure herein. For example, and without limitation, the singlevariable domains of the invention can be linked to a suitable carrier orsolid support so as to provide a medium than can be used in a mannerknown per se to purify members of the Notch signalling pathway fromcompositions and preparations comprising the same. Derivatives of thesingle variable domains of the invention that comprise a suitabledetectable label can also be used as markers to determine (qualitativelyor quantitatively) the presence of members of the Notch signallingpathway in a composition or preparation or as a marker to selectivelydetect the presence of members of the Notch signalling pathway on thesurface of a cell or tissue (for example, in combination with suitablecell sorting techniques).

The invention will now be further described by means of the followingnon-limiting examples and figures:

FIGURES

FIG. 1: The results in FIG. 1 show that most Nanobodies of Table B-1were able to bind to human Notch-1 in a concentration dependent manner.

FIG. 2: Binding assay of Nanobodies selected for Jagged-1. AllNanobodies were able to bind in a concentration dependent matter. Anirrelevant Nanobody is not able to bind to Jagged-1.

FIG. 3: Competition assay of Nanobodies selected for Jagged-1. Nanobody178C4 is able to compete for Jagged-1 binding to Notch-1 in aconcentration dependent matter. An irrelevant Nanobody is not able tocompete for Jagged-1 binding to Notch-1. Non-biotinylated Jagged-1 isalso able to compete for biot-Jagged-1 binding to Notch-1.

FIG. 4: Binding assay of Nanobodies selected for DLL4. All Nanobodiesare able to bind in a concentration dependent matter. An irrelevantNanobody is not able to bind to DLL4.

FIG. 5: Competition assay of Nanobodies selected for DLL4. SomeNanobodies are able to compete for DLL4 to Notch-1 in a concentrationdependent matter. An irrelevant Nanobody is not able to compete for DLL4binding to Notch-1.

FIG. 6: Binding assay to CD36-Fc (FIG. 6 a), human Notch-2 (FIG. 6 b),human Notch-1 (FIG. 6 c), and human Notch-3 (FIG. 6 d).

EXPERIMENTAL PART Example 1 Generation of Nanobodies SpecificallyBinding to human Notch-1 1.1. Animal Immunizations

Two llamas—lama glama (designated “Llama 161” and “Llama 166”) wereimmunized by intramuscular injection in the neck at 2 different spotswith 2 boosts of antigen cocktail preparations comprising 40 microgramof recombinant human Notch-1/Fc Chimera (R&D Systems Cat No 3647-TK) atday 0 and day 7 and 4 boosts of the same cocktail comprising 20microgram of recombinant human Notch-1/Fc Chimera at day 14, 21, 28 and35. The antigen cocktail was prepared by a) mixing above amounts ofrecombinant human Notch-1/Fc Chimera in a total of 2 ml PBS; and b)adding this mixture very slowly to 2.5 ml of the adjuvants Stimune(Stimune, previously known as Specol; Cedi-diagnostics, Lelystad, TheNetherlands); and c) mixing vigorously for 1 minute. Blood is collectedfrom these animals 4 and 8 days after boost 6. In addition,approximately 1 g of lymph node is collected from each animal 5 daysafter boost 6.

1.2. Library Construction

The library was prepared according to a standard protocol known to theskilled person in the art. In short, peripheral blood mononuclear cellsare prepared from blood samples using Ficoll-Hypaque according to themanufacturer's instructions. Next, total RNA was extracted from thesecells and lymph node tissue and used as starting material for RT-PCR toamplify VHH fragments encoding gene fragments. Then, a ligation wasprepared between VHH fragments prepared from an immunized animal (330 ngcDNA per library) and pAX50 vector digested with BstEII/SfiI/XhoI(pAX50, an expression vector, is used that is derived from pUC119 whichcontains the LacZ promoter, a coliphage pIII protein coding sequence, aresistance gene for ampicillin or carbenicillin, a multicloning site andthe gen3 leader sequence. In frame with the Nanobody coding sequence,the vector codes for a C-terminal c-myc tag and a His6× tag). Then,electrocompetent E. coli TG1 cells were transformed with ligated vectorscontaining the various VHH sequences by electroporation, grown atsuitable conditions, prepared for storage. So obtained librariescomprising phage particles were stored at −80 degrees Celsius.

1.3. Selections of Human Notch-1 Binding Nanobodies 1^(st) Round ofSelection:

The above prepared library was rescued, i.e. the above prepared phageparticles containing the VHH sequences were released upon maturation ofthe TG1 E. coli cells by infection with helper phage VCSM13 (Stratagene)with a multiplicity of infection of 10 (i.e. each bacterial cell isinfected with no more than 10 helper phage particles. Rescued phagelibraries designated as “library 161” (derived from “Llama 161”) and“library 166” (derived from “Llama 166”) were used for selections onrecombinant human Notch-1/Fc Chimera (R&D Systems Cat No3647-TK)—hereinafter “Notch-1/Fc”. Notch-1/Fc was immobilized directlyon Maxisorp 96 well microtiter plates (Nunc) at 5 microgram/ml and 0.5microgram/ml. In addition, a control without Notch-1/Fc was treated inparallel to the Notch/Fc incubated wells. To minimize the number ofphage binding to the Fc portion of Notch-1/Fc the phage waspre-incubated with 150 microgram/ml of human IgG for 1 h at roomtemperature (hereinafter “RT”). Following incubation with the rescuedphage libraries 161 and 166 (100 microliter of 10 microliter library in2% marvel PBS for 2 h at RT), extensive washing (PBS in 0.05% Tween 20),bound phage was eluted aspecific with 1 mg/ml freshly prepared trypsinin PBS. The eluted phages of library 161 and 166 were amplified and the5 microgram/ml Notch-1/Fc fractions of both libraries pooled and appliedto a second round of selection on 5 microgram/ml, 0.5 microgram/ml, 0microgram/ml (control) immobilized Notch-1/Fc. To minimize the number ofphage binding to the Fc-portion of Notch-1/Fc the phage is pre-incubatedwith 150 microgram/ml human IgG. Individual colonies obtained from theeluted phage pools (obtained from all 4 conditions, i.e. both librariesand both concentrations of Notch-1/Fc) were grown and induced with IPTGfor Nanobodies-expression and extraction (periplasmic extracts)according to standard methods.

1.4. Screening for Human Notch-1 Binding Nanobodies

In order to identify Nanobodies with binding specificity to humanNotch-1, individual clones picked from above selected Nanobodies arefurther tested in an ELISA binding assay setup. In short, 2 microgram/mlof human Notch-1/Fc was immobilized on Maxisorp ELISA plates (Nunc) inPBS over night (O/N) at 4 degrees Celsius (4° C.). After washing theplate was blocked using 4% Marvel skimmed milk in PBS for 30 minutes onshaker. A concentration series of Nanobody (1 to 1000 nM) was added in100 microliter 2% Marvell/PBS and incubated for 1 h on shaker. Afterwashing human Notch-1 binding Nanobodies were detected with 100microliter 1:5000 Mouse-anti-MycTag in 2% Marvell/PBS and 100 microliter1:5000 DAMPO in 2% Marvell/PBS for 1 h on shaker. PO was detected with100 microliter OPD and stopped with 50 microliter H2SO4. Quantificationwas performed at 490 nm in a plate reader. The results in FIG. 1 showthat most Nanobodies of Table B-1 were able to bind to human Notch-1 ina concentration dependent manner.

TABLE B-1 Selected Nanobodies against human Notch-1. SEQ ID Name NO:Sequence >180A4 339 EVQLVESGGGLVQPGGSLRLSCAASGRTSSPIAMGWFRQAPGKEREFVSGITWSGAYTHYANSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCTTATNSTTGYDYWGQGTQVTVSS >180B1 340EVQLVESGGDLVQPGGSLRLSCAASGTVFSNNDMGWSRQAPGKERELVASFTSGGNTVYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDLLRGKLYWGQGTQVTVSS >180B3 341EVQLVESGGDLVQPGGSLRLSCAASGTVFSNNDMGWSRQAPGKERELVASFTSGGNTVYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDLLRGKLYWGQGTQVTVSS >180C1 342EVQLVESGGDLVQPGGSLRLSCAASRTVFSISDMGWSRQAPGKERELVASISSDNYTVYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS >180C7 343EVQLVESGGGLVQAGGSLRLSCAASGGIFGINAVAWYRQAPGKERDWVALIVGEAITRYTDSVSGRFTISRDNAKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS >180F8 344EVQLVESGGDLVQPGGSLRLSCTTSGTVFSINDMGWSRQAPGKGRELVASISSEGTTIYADAAKGRFTISRDNSKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS >180F4 345EVQLVESGGGLVQAGDSLRLSCAVSGGSFSSYTMGWVRQAPGKEREAVASIWRSGGNTYYADSVKGRFTVSRDNAKHTVYLQMNSLKPEDTAVYYCAAASFAPGSRGYDYWGQGTQVTVSS >180F5 346EVQLVESGGGLVQPGGSLRLSCAASGRTSSPIAMGWFRQAPGKEREFVSGITWSGAYTHYANSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCTTATNST TGYDYWGQGTQVTVSSAbove set of selected single variable domains (Nanobodies™) consists of5 families:

Family 1: 180B1, 180B3, 180F10 Family 2: 180A4 Family 3: 180F4 Family 4:180C11 Family 5: 180C1, 180F8, 18007 1.5. Selection for Notch-1 SpecificBinders

In order to select for Notch-1 binders that do not cross-react withNotch-2 and Notch-3 second round selections were performed withcounterselection in solution with Notch-2 and Notch-3. Binding assayswere performed. In short: Maxisorp plate was coated with 100 ul 2 ug/mlof rhNotch-1 or Notch-2 (R&D systems) in PBS O/N at 4° C. After washingthe plate was blocked with 4% MPBS for 30 minutes hour on shaker. Ten ulof Nanobody from the periplasmic extract was added in 100 ul 2%Marvell/PBS and incubate for 1 hour on shaker. After washing Nanobodieswere detected with 100 ul 1:5000 Mouse-anti-MycTag in 2% Marvell/PBS and100 ul 1:5000 DAMPO in 2% Marvell/PBS for 1 hour on shaker. PO wasdetected with 100 ul OPD and stopped with 50 ul H2SO4. Quantificationwas performed at 490 nm in a plate reader. The results showed that fromthe 96 clones tested 85% was able to bind to Notch-1 and only 2% wascross-reactive for Notch-2, indicating that most selected clones werespecific for Notch-1.

Example 2 Generation of Nanobodies Specifically Binding to Human Notch-22.1. Immunizations

Two llamas were immunized with 6 boosts of R&D Systems Cat #3735-NT-050,which is the ectodomain of human Notch-2, according to standardprotocols, e.g. as above.

2.2. Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA extracted was extracted from these cells and used as startingmaterial for RT-PCR to amplify Nanobody encoding gene fragments. Thesefragments were cloned into phagemid vector pAX50. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored after filtersterilization at 4° C. for further use.

2.3. Selection

Two rounds of phage display selection were performed according tostandard protocols, see above. Binding assays (screening) were performedas above. In short: Maxisorp plate was coated with 100 ul 2 ug/ml ofNotch-2 (R&D systems) or with total human IgG in PBS O/N at 4° C. Afterwashing the plate 4× with PBS the plate was blocked with 4% Marvel inPBS for 30 minutes on shaker. 10 ul of periplasmic extract of Nanobodywas added in 100 ul 2% Marvell/PBS and incubate for 1 hour on shaker.After washing the plate 4× with PBS the Nanobodies were detected with100 ul 1:5000 Mouse-anti-MycTag in 2% Marvell/PBS for 1 hour and 100 ul1:5000 DAMPO in 2% Marvell/PBS for 1 hour. After washing the DAMPO wasdetected with 100 ul OPD, stopped with 50 ul H2SO4 and quantificationwas performed at 490 nm in a plate reader. Results showed that the 90%of the Nanobodies bind to the Notch-2-Fc fusion (see below Table). Twoof the clones were shown to bind to IgG as well. 181C10, 181C11 and181D11 belong to one family

TABLE B-2 Selected Nanobodies against human Notch-2. SEQ ID Name NO:Sequence >181G4 348 EVQLVESGGGLVQAGGSLRLSCAASGSIFSITEMDWYRQAPGKQREWVAGETSDGSTNYADSVKGRFTISRDNANNAVYLQMNRLKPEDTAVYHCAASLRNSGSNVEGRYWGQGTQVTVSS >181B4 349EVQLVESGGGLVQPGGSLRLSCAASGRINSMNWYHQAPGKEREWVASITSSGTAIYADSVKGRITISRDNAKNTVYLQMNSLKTEDTGVYYCAANLQNARGSYYGQ GTQVTVSS >181D4350 EVQLVESGGGLVQPGGSLRLSCAASGFTFGDYAMNWVRQAPGKGLEWVSSISAGGYSTTYADSVKGRFTISRDNAKNTLYLQMNSLNPEDTAVYYCARGDWRYGSRGQGTQVTVSS >181E4 351EVQLVESGGGLVQAGGSLRLSCAASGSMSSINAMRWYRQASGKQREPVAEITSEGTIIYADSVKGRFTTSRDNAKNTVYLQMNSLKPEDTGVYYCAADDGARGSY YGQGTQVTVSS >181F4352 EVQLVESGGALVQAGGSLRLSCVASGSSFSINDMDWYRQAPGKTREWVAGINEYGGRNYANSVKDRFTISTDNAKNTVYLQMNSLKPEDTGVYYCAATLAKGGGRYWGQGTPVTVSS >181H4 353EVKLVESGGGLVQPGGSLRLSCAASGSIFRFNGVDWYRQAPGAEREWVAGFGSGGTTNYADSVKGRFIVSRDNAENTVFLQMNSLKPEDSAVYFCAASIEGVSGRYYGQGTQVTVSS >181A5 354EVQLVESGGGLVQAGGSLRLSCVVSGSILSINTMDWYRQAPGNEREWVGGITDGGRSNYADSVKDRFTIYRANAKNTVYLQMNSLKPEDTAVYYCAADLRGGIATTGRYWGQGTQVTVSS >181C10 355EVQLVESGGGLVQAGGSLRLSCAASGMTTIGPMGWYRQGPGKQRELVARITGGGSTNYVDSAKGRFTISRDNAKNAVYLQMNNLKLDDTAVYYCNAYGSGSDYLPIDYWGQGTQVTVSS >181C11 356EVQLVESGGGLVQAGGSLRLSCAASGMTTIGPMGWYRQGPGKQRELVARITGGGSTNYVDSAKGRFTISRDNAKNMGYLQMNNLKLDDTAIYYCYAYGSGSDYLPTDYWGQGTQVTVSS >181D11 357EVQLVESGGGLVQAGGSLRLSCAASGMTTIGPMGWYRQGPGKQRELVARITGGGSTNYVDSAKGRFTISRHNAKNMVYLQMNNLKLDDTAVYYCNAYGSGSDYL PMDYWGQGTQVTVSS

Example 3 Generation of Nanobodies Specifically Binding to Jagged-1 3.1.Immunizations

Two llamas were immunized with 6 boosts of R&D Systems Cat #599-JG,which is the ectodomain of rat Jagged-1, according to standardprotocols.

3.2. Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA extracted was extracted from these cells and used as startingmaterial for RT-PCR to amplify Nanobody encoding gene fragments. Thesefragments were cloned into phagemid vector pAX50. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored after filtersterilization at 4° C. for further use.

3.3. Selection

Two rounds of phage display selection were performed similarly asdescribed above. Binding assays were performed. In short: Maxisorp platewas coated with 100 ul 2 ug/ml of rrJagged-1 (R&D systems) in PBS O/N at4 C. After washing the plate 4× with PBS the plate was blocked with 4%Marvel in PBS for 30 minutes on shaker. A concentration series ofNanobody (1-1000 nM) was added in 100 ul 2% Marvell/PBS and incubate for1 hour on shaker. After washing the plate 4× with PBS the Nanobodieswere detected with 100 ul 1:5000 Mouse-anti-MycTag in 2% Marvell/PBS for1 hour and 100 ul 1:5000 DAMPO in 2% Marvell/PBS for 1 hour. Afterwashing the DAMPO was detected with 100 ul OPD, stopped with 50 ul H2SO4and quantification was performed at 490 nm in a plate reader. Resultsare shown in FIG. 2 and show that four Nanobodies (Table B-3) bind torat Jagged-1, where 178C4 seems to have a lower affinity for Jagged-1compared to the others.

TABLE B-3 Selected Nanobodies against rat Jagged-1. SEQ ID Name NO:Sequence >178C4 335 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCMRGRDGSTYYADSVNGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAHTDFACYIGYYSDYDPHDYWGQGTQVTVSS >178A7 336EVQLVESGGGLVQPGGSLRLSCATSGVIFSTAAMGWYRQAPGKSRVLVARMFSDGRTIYAESVKRRFTISRDNAKNTVYLQMNSLEPEDTAVYYCNALQFGAVYW GQGTQVTVSS >178D9337 EVQLVKSGGGLVQPGGSLRLSCAASGSIVSANLIGWYRQAPGKQREGVAFITSGGAINYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAIYYCNLRQLGNVYWA QGTQVTVSS >178C11338 EVQLVESGGGLVQPGGSLRLSCATSGVIFSTAAMGWYRQAPGKSRVLVARMFSDGRTIYAESVKRRFTISRDNAKNTVYLQMNSLEPEDTAVYYCNALQFGAVYW GQGTQVTVSSAbove selected single variable domains (Nanobodies™) consists of 3families:

3 Families:

Family 1: 1 competitive binder: 178C4Family 2: 1 binder: 178D9Family 3: 2 binders: 178A7 and 178C11

3.4. Competition Assay

Competition ELISA assay was performed to check the competition ofJagged-1 binding to human Notch-1 by the Nanobodies. In short: Maxisorpplate was coated with 100 ul 5 ug/ml of rhNotch-1/hFc (R&D systems) inPBS O/N at 4° C. After washing the plate 4× with PBS the plate wasblocked with 4% Marvel PBS for 30 minutes hour on shaker. Aconcentration series of Nanobody (1-1000 nM) and 0.3 ug/ml biotinylatedrrJagged-1 (R&D systems, biotinylated in-house) was added in 100 ul 2%Marvell/PBS and incubated for 1 hour on shaker. (0.3 ug/ml biotinylatedrrJagged-1˜2.1 nM). After washing the biotin was detected with 100 ul1:5000 streptavidin-PO in 2% Marvell/PBS for 1 hour on shaker. PO wasdetected with 100 ul OPD and stopped with 50 ul H2SO4. Quantificationwas performed at 490 nm in a plate reader. The results are shown in FIG.3 and show that 178C4 is able to compete for Jagged-1 binding to Notch-1in a concentration dependent matter. The competition of 178C4 iscomparable to the competition by adding non-biotinylated Jagged-1(cold-jagged1).

Example 4 Generation of Nanobodies Specifically Binding to Human DLL44.1. Immunizations

Two llamas were immunized with 6 boosts of R&D Systems Cat #1506-D4-050,which is the ectodomain of human DLL4, according to standard protocols.

4.2. Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA extracted was extracted from these cells and used as startingmaterial for RT-PCR to amplify Nanobody encoding gene fragments. Thesefragments were cloned into phagemid vector pAX50. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored after filtersterilization at 4° C. for further use.

4.3. Selection

Two rounds of phage display selection were performed similarly asdescribed above. Binding assays were performed. In short: Maxisorp platewas coated with 100 ul 5 ug/ml of rhDLL4 (R&D systems) in PBS O/N at 4°C. After washing the plate was blocked with 4% MPBS for 30 minutes houron shaker. A concentration series of Nanobody (1-1000 nM) was added in100 ul 2% Marvell/PBS and incubate for 1 hour on shaker. Nanobody wasdetected with 100 ul 1:5000 Mouse-anti-MycTag (9E10) in 2% Marvell/PBSand with 100 ul 1:5000 DAMPO in 2% Marvell/PBS for 1 hour on shaker.After washing PO was detected with 100 ul OPD and stopped with 50 ulH2SO4. Quantification was performed at 490 nm in a plate reader. Resultsshow in FIG. 4 that all Nanobodies show a concentration dependentbinding to DLL4, where 179F7 shows only little binding at the highestconcentration.

TABLE B-4 Selected Nanobodies against human DLL4. SEQ ID Name NO:Sequence >179E6 329 EVQLVESGGGLVQAGGSLRLSCIASGDTSEIYDMGWFRQAPGKEREFVSSIHWRGRGTNYTDSVEGRFTISKDNAKNMVYLQMNSLKPEDTAVYYCAATRLIVYTPTGFRQYFDWGQGAQVTVSS >179H9 330EVQLVESGGGLVQAGGSLRLSCAASG??F?SDVMGWFRRAPGKEREFVASITTTGNEYYEDSLKGRFTVSRDIAENTMYLEMTNLKPEDTAEYSCAGRLLGSTIRSHEYRYWGQGTQVTVSS >179A10 331EVQLVESGGGLVQAGGSLRLSCVASGDTSEIYDMGWFRQAPGKEREFVSSIHWGGRGTNYTDSVKGRFTISKDNAKNMVHLQMNSLKPEDTAVYYCAATRLIVYTPTGFRQYFDWGQGTQVTVSS >179D12 332EVQLVESGGGLVQAGGSLRLSCVASGDTSEIYDMGWFRQAPGKEREFVSSIHWGGRGTNYTDSVKGRFTISKDNAKNMVYLQMNSLKPEDTAVYYCAATRLIVYTPTGFRQYFDWGQGTQVTVSS >179F6 333EVQLVESGGGLVQAGGSLRLSCAASGSDFSGFLLGWYRQPPGKQRELIAQTTDGGRTNYGDSVKGRFTISRDNAKNTWYLQMDSLKPEDTGVYYCNTFPLTSWG QGTQVTVSS >179F7334 EVQLVESGGGLVQSGGSLSLSCAASGSVLRINDMGWYRQAPGKTREMVATITRSGVLNYTDSVKGRFTVSRDDAKNTVYLQMSSLKPEDTAVYSCFANIVIRSSGY FNRYWGQGTLVTVSSAbove selected single variable domains (Nanobodies™) consists of 4families:

4 Families

Family 1: competitive binders: 179A10, 179D12, 179E6Family 2: competitive binder: 179H9Family 3: competitive binder: 179F6Family 4: binder: 179F7

4.4. Competition

Competition ELISA assay was performed to check the competition of DLL4binding to human Notch-1 by the Nanobodies. In short: Maxisorp plate wascoated with 100 ul 5 ug/ml of rhDLL4 (R&D systems) in PBS O/N at 4° C.After washing the plate 4× with PBS the plate was blocked with 4% MarvelPBS for 30 minutes hour on shaker. A concentration series of Nanobody(1-1000 nM) and 0.2 ug/ml rhNotch-1/hFc (R&D systems) was added in 100ul 2% Marvell/PBS and incubated for 1 hour on shaker. After washing theNotch-Fc binding was detected with 100 ul 1:5000 anti-hFc-HRP (Jackson)in 2% Marvell/PBS for 1 hour on shaker. PO was detected with 100 ul OPDand stopped with 50 ul H2SO4. Quantification was performed at 490 nm ina plate reader. The results show in FIG. 5 that all Nanobodies, exceptfor 179F7, are able to compete for DLL4 binding to Notch-1 in aconcentration dependent matter. The competition of the Nanobodies iscomparable to the competition by adding non-biotinylated Jagged-1(cold-jagged1).

Example 5 Most Nanobodies Bind Only to One Member of the Notch Family

Various Nanobodies were generated as above against human Notch-1,Notch-2, and Notch-3 (see Table B-5).

TABLE B-5 Selected Nanobodies against human Notch-1, Notch-2, andNotch-3 Name of SEQ ID NO: Target Nanobody Amino acid sequence 358 Human181B3 EVQLVESGGGLVQAGGSLRLSCVVSGSILSINTMD Notch-2WYRQAPGKQREWFAGLSDGGRANYADSVKDRFT IARDNAKNTVYLQMNSLKPGDTAIYYCAADLWDRGATTGRYWGQGTQVTVSS 359 Human 181D2 EVQLVESGGGLVQPGGSLRLSCAASGRINSMNWYNotch-2 RQAPGKEREWVASITSSGTTIYADSVKGRITISEDNAKNTVYLQMNSLKTEDTGVYYCAANLRNARGSY YGQGTQVTVSS 360 Human 181D3EVQLVESGGGLVQAGGSLRLSCAASTNVSSFNTM Notch-2SWYRQSPRKQREWVATITDGDNTHYADSVKGRFT TSRDNAKNLLYLQMNSLKPEDTAVYYCTGWRATSGGSYGQGTQVTVSS 361 Human 181E3 EVQLVESGGGLVQPGGSLRLSCAASGFTFGDYAMNotch-2 NWVRQAPGKGLEWVSSISAGGYSTTYADSVKGRFTISRDNAKNTLYLQMNSLNPEDTAVYYCARGDW RYGSRGQGTQVTVSS 362 Human 50C4EVQLVESGGDLVQPGGSLRLSCAASGFTFSTYYMS Notch-3WVRQAPGKGLEWVSGIYSDGSNTYYADSVKGRF TISRDNAKNTLYLQMNSLKSEDTAVYYCTRPVVVAGSPVSYEYDYIGQGTQVTVSS 363 Human 50H9EVQLVESGGDLVQPGGSLRLSCAASGFTFSTYYMS Notch-3WVRQAPGKGLEWVSGIYSDGSNTYYADSVKGRF TISRDNAKNTLYLQMNSLKSEDTAVYYCTRPVVVAGSPVSYEYDYIGQGTQVTVSS 364 Human 180F8EVQLVESGGDLVQPGGSLRLSCTTSGTVFSINDMG Notch-1WSRQAPGKGRELVASISSEGTTIYADAAKGRFTISR DNSKNTVYLEMNSLKPDDTAVYYCRVVDPLRGKLYWGQGTQVTVSS 365 Negative 4H2 EVQLVESGGGLVQAGGSLRLSCATSGFTFSRFDMScontrol WFRQAPGKQREFIATIFSGGDTDYIDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCCPLGIEYAW GQGTQVTVSSIn order to test whether Nanobodies with binding specificity to humanNotch-1, -2 or -3 have cross-reactivity to other members of the Notchfamily, individual clones picked from above selected Nanobodies arefurther tested in an ELISA binding assay setup. In short, 2 microgram/mlof human Notch-1, Notch-2, Notch-3 and CD36-Fc was immobilized onMaxisorp ELISA plates (Nunc) in marvell PBST for 1 h at 4 degreesCelsius (4° C.). After washing, the plate was blocked using 4% Marvelskimmed milk in PBS for 30 minutes on shaker. A concentration series ofthe Nanobodies in Table B-5 (25 pM to 2000 nM) was added in 100microliter 2% Marvell/PBS and incubated for 1 h on shaker. After washingthe Nanobodies were detected with 100 microliter 1:2500 R-anti-VHH(1:2500) in 2% Marvell/PBS and 100 microliter 1:5000 DAMPO in 2%Marvell/PBS for 1 h on shaker. PO was detected with 100 microliter OPDand stopped with 50 microliter H2SO4. Quantification was performed at490 nm in a plate reader. The results in FIG. 6 show that:

-   -   None of the Notch clones binds to Fc (FIG. 6 a)    -   50H9 (Notch-3 binder) shows some crossreactivity with Notch-1        (FIGS. 6 c and 6 d)    -   All other clones seem to be specific FIGS. 6 b, 6 c, 6 d    -   Binding (IC50) is in the low nanomolar range

Example 6 List of General In Vitro, Cell-Based or In Vivo Assays

In vitro binding assays: ELISA, Biacore

In vivo binding assay: Flow cytometry

In vivo functional assay: Mouse models for several diseases where Notchis involved have been developed (Gridley T., 2003, Human MolecularGenetics, Vol 12, Review Issue 1, R9-R13)

Example 7 List of Target Proteins of the Invention (Links to Nucleic andAmino Acid Sequence)

Sequence of human Sequences from various members of the Notch speciesfound e.g. on signalling pathwayhttp://www.ncbi.nlm.nih.gov/sites/entrez Human Notch-1 AAG33848 HumanNotch-2 AAA36377 Human Notch-3 CAA55955 Human Notch-4 Q99466 HumanJagged-1 AAC52020 Human Jagged-2 AAB61285 Human DLL1 AAQ89251 Human DLL3AAF62542 Human DLL4 AAQ89253

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

PREFERRED ASPECTS

1. A single variable domain that specifically binds to at least onemember of the Notch pathway.

2. The single variable domain according to aspect 1, wherein the memberof the Notch pathway is selected from the group consisting of the humanmembers of the Notch pathway.

3. The single variable domain according to aspect 1, wherein the memberof the Notch pathway is selected from the group consisting of the Notchreceptors and Notch ligands.

4. The single variable domain according to aspect 1, wherein the memberof the Notch pathway is selected from the group consisting of the humanNotch receptors and human Notch ligands.

5. The single variable domain according to aspect 1, wherein the memberof the Notch pathway is selected from the group consisting of Jagged-1,Jagged-2, DLL1, DLL3, DLL4, Notch-1, Notch-2, Notch-3, and Notch-4.

6. The single variable domain according to aspect 1, wherein the memberof the Notch pathway is selected from the group consisting of humanJagged-1, human Jagged-2, human DLL1, human DLL3, human DLL4, humanNotch-1, human Notch-2, human Notch-3, and human Notch-4.

7. The single variable domain according to aspect 1, wherein the memberof the Notch pathway is selected from the group consisting of humanJagged-1, human DLL4, human Notch-1 and human Notch-2.

8. The single variable domain according to aspect 1, wherein the singlevariable domain additionally blocks the interaction between at least onemember of the Notch ligands with at least one other member of the Notchreceptors.

9. The single variable domain according to aspect 1, wherein the singlevariable domain additionally blocks the interaction between at least onemember of the group consisting of Notch ligands and Notch receptors withat least one single variable domain with sequences having SEQ ID NO: 329to 364.

10. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 329 to 364.

11. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

12. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

13. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

14. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

15. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 329 to 364; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

16. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

17. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

18. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

19. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

20. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 329 to 364; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

21. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

22. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences SEQ ID NO: 329 to 364 wherein upto 8 amino acid residues are replaced by naturally occurring amino acidsand wherein said replaced amino acids are located within the frameworkregions; and wherein said selected single variable domain from group a)and b) binds to at least one member of the Notch signalling pathway witha dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

23. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

24. The single variable domain according to aspect 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch signallingpathway with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

25. A single variable domain that specifically binds to at least onemember of the Notch receptors.

26. The single variable domain according to aspect 25, wherein themember of the Notch receptors is selected from the group consisting ofthe human members of the Notch receptors.

27. The single variable domain according to aspect 25 wherein the memberof the Notch receptors is selected from the group consisting of Notch-1,Notch-2, Notch-3, and Notch-4.

28. The single variable domain according to aspect 25 wherein the memberof the Notch receptors is selected from the group consisting of humanNotch-1, human Notch-2, human Notch-3, and human Notch-4.

29. The single variable domain according to aspect 25 wherein the memberof the Notch receptors is selected from the group consisting of humanNotch-1 and human Notch-2.

30. The single variable domain according to aspect 25, wherein thesingle variable domain additionally blocks the interaction between atleast one member of the Notch ligands with at least one other member ofthe Notch receptors.

31. The single variable domain according to aspect 25, wherein thesingle variable domain additionally blocks the interaction between atleast one member of the group consisting of Notch receptors with atleast one single variable domain with sequences having SEQ ID NO: 339 to364.

32. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 339 to 364.

33. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

34. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

35. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

36. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

37. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 339 to 364; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

38. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

39. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

40. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

41. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

42. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 339 to 364; and wherein said selected single variable domain fromgroup a) and b) binds to members of the Notch signalling pathway with adissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

43. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

44. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

45. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

46. The single variable domain according to aspect 25, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 364; andb) single variable domains with sequences having SEQ ID NO: 339 to 364,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch receptors witha dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

47. A single variable domain that specifically binds to at least onemember of the Notch ligands.

48. The single variable domain according to aspect 47, wherein themember of the Notch ligands is selected from the group consisting of thehuman members of the Notch ligands.

49. The single variable domain according to aspect 47, wherein themember of the Notch receptors is selected from the group consisting ofJagged-1, Jagged-2, DLL1, DLL3, DLL4.

50. The single variable domain according to aspect 47, wherein themember of the Notch receptors is selected from the group consisting ofhuman Jagged-1, human Jagged-2, human DLL1, human DLL3, and human DLL4.

51. The single variable domain according to aspect 47, wherein themember of the Notch receptors is selected from the group consisting ofhuman Jagged-1 and human DLL4.

52. The single variable domain according to aspect 47, wherein thesingle variable domain additionally blocks the interaction between atleast one member of the Notch ligands with at least one other member ofthe Notch receptors.

53. The single variable domain according to aspect 47, wherein thesingle variable domain additionally blocks the interaction between atleast one member of the group consisting of Notch ligands with at leastone single variable domain with sequences having SEQ ID NO: 329 to 338.

54. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 329 to 338.

55. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

56. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

57. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

58. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

59. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 329 to 338; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

60. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

61. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

62. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

63. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

64. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 329 to 338; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

65. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

66. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

67. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

68. The single variable domain according to aspect 47, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 338; andb) single variable domains with sequences having SEQ ID NO: 329 to 338,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to at least one member of the Notch ligands with adissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

69. A single variable domain that specifically binds to at least onemember of the DLL4 family.

70. The single variable domain according to aspect 69, wherein themember of the DLL4 family is selected from human DLL4.

71. The single variable domain according to aspect 69, wherein thesingle variable domain additionally blocks the interaction between humanDLL4 with at least one other member of the DLL4 family.

72. The single variable domain according to aspect 69, wherein thesingle variable domain additionally blocks the interaction between humanDLL4 with at least one single variable domain with sequences having SEQID NO: 329 to 334.

73. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 329 to 334.

74. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

75. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

76. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

77. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

78. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 329 to 334; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁷ to 10⁻¹² moles/liter or less.

79. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁷ to 10⁻¹² moles/liter or less.

80. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁷ to 10⁻¹² moles/liter or less.

81. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁷ to 10⁻¹² moles/liter or less.

82. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁷ to 10⁻¹² moles/liter or less.

83. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 329 to 334; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁸ to 10⁻¹² moles/liter or less.

84. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁸ to 10⁻¹² moles/liter or less.

85. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁸ to 10⁻¹² moles/liter or less.

86. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁸ to 10⁻¹² moles/liter or less.

87. The single variable domain according to aspect 69, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 329 to 334; andb) single variable domains with sequences having SEQ ID NO: 329 to 334,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human DLL4 with a dissociation constant (K_(D))of 10⁻⁸ to 10⁻¹² moles/liter or less.

88. A single variable domain that specifically binds to at least onemember of the Jagged-1 family.

89. The single variable domain according to aspect 88, wherein themember of the Jagged-1 family is selected from human Jagged-1.

90. The single variable domain according to aspect 88, wherein thesingle variable domain additionally blocks the interaction between humanJagged-1 with at least one other member of the Jagged-1 family.

91. The single variable domain according to aspect 88, wherein thesingle variable domain additionally blocks the interaction between humanJagged-1 with at least one single variable domain with sequences havingSEQ ID NO: 335 to 338.

92. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 335 to 338.

93. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

94. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

95. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

96. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

97. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 335 to 338; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

98. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

99. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

100. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

101. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

102. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 335 to 338; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

103. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

104. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

105. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

106. The single variable domain according to aspect 88, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 335 to 338; andb) single variable domains with sequences having SEQ ID NO: 335 to 338,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Jagged-1 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

107. A single variable domain that specifically binds to at least onemember of the Notch-1 family.

108. The single variable domain according to aspect 107, wherein themember of the Notch-1 family is selected from human Notch-1.

109. The single variable domain according to aspect 107, wherein thesingle variable domain additionally blocks the interaction between humanNotch-1 with at least one other member of the Notch ligands.

110. The single variable domain according to aspect 107, wherein thesingle variable domain additionally blocks the interaction between humanNotch-1 with at least one single variable domain with sequences havingSEQ ID NO: 339 to 347 and SEQ ID NO: 364.

111. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with 80% sequenceidentity to at least one sequence selected from the group consisting ofsingle variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364.

112. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 10 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions.

113. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 8 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions.

114. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 5 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions.

115. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 3 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions.

116. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with 80% sequenceidentity to at least one sequences selected from the group consisting ofsequences having SEQ ID NO: 339 to 347 and SEQ ID NO: 364; and whereinsaid selected single variable domain from group a) and b) binds to humanNotch-1 with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

117. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 10 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

118. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 8 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

119. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains sequences having SEQ IDNO: 339 to 347 and SEQ ID NO: 364, wherein up to 5 amino acid residuesare replaced by naturally occurring amino acids and wherein saidreplaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

120. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 3 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹²moles/liter or less.

121. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with 80% sequenceidentity to at least one sequences selected from the group consisting ofsequences having SEQ ID NO: 339 to 347 and SEQ ID NO: 364; and whereinsaid selected single variable domain from group a) and b) binds to humanNotch-1 with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

122. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 10 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

123. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 8 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

124. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 5 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

125. The single variable domain according to aspect 107, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 339 to 347 andSEQ ID NO: 364; and b) single variable domains with sequences having SEQID NO: 339 to 347 and SEQ ID NO: 364, wherein up to 3 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions; andwherein said selected single variable domain from group a) and b) bindsto human Notch-1 with a dissociation constant (K_(D)) of 10⁻⁸ to 10⁻¹²moles/liter or less.

126. A single variable domain that specifically binds to at least onemember of the Notch-2 family.

127. The single variable domain according to aspect 126, wherein themember of the Notch-2 family is selected from human Notch-2.

128. The single variable domain according to aspect 126, wherein thesingle variable domain additionally blocks the interaction between humanNotch-2 with at least one other member of the Notch ligands.

129. The single variable domain according to aspect 126, wherein thesingle variable domain additionally blocks the interaction between humanNotch-2 with at least one single variable domain with sequences havingSEQ ID NO: 348 to 361.

130. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 348 to 361.

131. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

132. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

133. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

134. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

135. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 348 to 361; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

136. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

137. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

138. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

139. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

140. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 348 to 361; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

141. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

142. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

143. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

144. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 348 to 361; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

145. A single variable domain that specifically binds to Jagged-2.

146. The single variable domain according to aspect 145, wherein themember of the Jagged-2 family is selected from the group consisting ofthe human Jagged-2.

147. The single variable domain according to aspect 145, wherein thesingle variable domain additionally blocks the interaction between humanJagged-2 with at least one other member of the Notch receptors.

148. A single variable domain that specifically binds to DLL1.

149. The single variable domain according to aspect 148, wherein themember of the DLL1 family is selected from the group consisting of thehuman DLL1.

150. The single variable domain according to aspect 148, wherein thesingle variable domain additionally blocks the interaction between humanDLL1 with at least one other member of the Notch receptors.

151. A single variable domain that specifically binds to DLL3.

152. The single variable domain according to aspect 151, wherein themember of the DLL3 family is selected from the group consisting of thehuman DLL3.

153. The single variable domain according to aspect 151, wherein thesingle variable domain additionally blocks the interaction between humanDLL3 with at least one other member of the Notch receptors.

154. A single variable domain that specifically binds to at least onemember of the Notch-3 family.

155. The single variable domain according to aspect 154, wherein themember of the Notch-2 family is selected from human Notch-3.

156. The single variable domain according to aspect 154, wherein thesingle variable domain additionally blocks the interaction between humanNotch-3 with at least one other member of the Notch ligands.

157. The single variable domain according to aspect 154, wherein thesingle variable domain additionally blocks the interaction between humanNotch-3 with at least one single variable domain with sequences havingSEQ ID NO: 362 to 363.

158. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 362 to 363.

159. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

160. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

161. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

162. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.

163. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 362 to 363; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

164. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 348 to 361,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

165. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

166. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

167. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.

168. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with 80% sequence identity to at least onesequences selected from the group consisting of sequences having SEQ IDNO: 362 to 363; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

169. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 10 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-2 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

170. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

171. The single variable domain according to aspect 126, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 5 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

172. The single variable domain according to aspect 154, wherein thesingle variable domain is selected from the group consisting of a)single variable domains with sequences having SEQ ID NO: 362 to 363; andb) single variable domains with sequences having SEQ ID NO: 362 to 363,wherein up to 3 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions; and wherein said selected single variable domain fromgroup a) and b) binds to human Notch-3 with a dissociation constant(K_(D)) of 10⁻⁸ to 10⁻¹² moles/liter or less.

173. A single variable domain that specifically binds to Notch-4.

174. The single variable domain according to aspect 173, wherein themember of the Notch-3 family is selected from the group consisting ofthe human Notch-4.

175. The single variable domain according to aspect 173, wherein thesingle variable domain additionally blocks the interaction between humanNotch-4 with at least one other member of the Notch ligands.

176. Single variable domain with sequence having SEQ ID NO: 329.

177. Single variable domain with sequence having SEQ ID NO: 330.

178. Single variable domain with sequence having SEQ ID NO: 331.

179. Single variable domain with sequence having SEQ ID NO: 332.

180. Single variable domain with sequence having SEQ ID NO: 333.

181. Single variable domain with sequence having SEQ ID NO: 334.

182. Single variable domain with sequence having SEQ ID NO: 335.

183. Single variable domain with sequence having SEQ ID NO: 336.

184. Single variable domain with sequence having SEQ ID NO: 337.

185. Single variable domain with sequence having SEQ ID NO: 338.

186. Single variable domain with sequence having SEQ ID NO: 339.

187. Single variable domain with sequence having SEQ ID NO: 340.

188. Single variable domain with sequence having SEQ ID NO: 341.

189. Single variable domain with sequence having SEQ ID NO: 342.

190. Single variable domain with sequence having SEQ ID NO: 343.

191. Single variable domain with sequence having SEQ ID NO: 344.

192. Single variable domain with sequence having SEQ ID NO: 345.

193. Single variable domain with sequence having SEQ ID NO: 346.

194. Single variable domain with sequence having SEQ ID NO: 347.

195. Single variable domain with sequence having SEQ ID NO: 348.

196. Single variable domain with sequence having SEQ ID NO: 349.

197. Single variable domain with sequence having SEQ ID NO: 350.

198. Single variable domain with sequence having SEQ ID NO: 351.

199. Single variable domain with sequence having SEQ ID NO: 352.

200. Single variable domain with sequence having SEQ ID NO: 353.

201. Single variable domain with sequence having SEQ ID NO: 354.

202. Single variable domain with sequence having SEQ ID NO: 355.

203. Single variable domain with sequence having SEQ ID NO: 356.

204. Single variable domain with sequence having SEQ ID NO: 356.

205. Single variable domain with sequence having SEQ ID NO: 357.

206. Single variable domain with sequence having SEQ ID NO: 358.

207. Single variable domain with sequence having SEQ ID NO: 359.

208. Single variable domain with sequence having SEQ ID NO: 360.

209. Single variable domain with sequence having SEQ ID NO: 361.

210. Single variable domain with sequence having SEQ ID NO: 362.

211. Single variable domain with sequence having SEQ ID NO: 363.

212. Single variable domain with sequence having SEQ ID NO: 364.

213. Single variable domain with sequence having SEQ ID NO: 365.

214. A construct comprising at least one single variable domain ofaspects 1 to 24.

215. A construct comprising at least one single variable domainaccording to aspects 25 to 46.

216. A construct comprising at least one single variable domain ofaspects 47 to 68.

217. A construct comprising at least one single variable domain ofaspects 69 to 87.

218. A construct comprising at least one single variable domain ofaspects 88 to 106.

219. A construct comprising at least one single variable domain ofaspects 107 to 125.

220. A construct comprising at least one single variable domain ofaspects 126 to 144.

221. A construct comprising at least one single variable domain ofaspects 145 to 147.

222. A construct comprising at least one single variable domain ofaspects 148 to 150.

223. A construct comprising at least one single variable domain ofaspects 151 to 153.

224. A construct comprising at least one single variable domain ofaspects 154 to 172.

225. A construct comprising at least one single variable domain ofaspects 173 to 175.

226. A construct comprising at least one single variable domain ofaspects 107 to 125.

227. A construct comprising at least one single variable domain ofaspect 176.

228. A construct comprising at least one single variable domain ofaspect 177.

229. A construct comprising at least one single variable domain ofaspect 178.

230. A construct comprising at least one single variable domain ofaspect 179.

231. A construct comprising at least one single variable domain ofaspect 180.

232. A construct comprising at least one single variable domain ofaspect 181.

233. A construct comprising at least one single variable domain ofaspect 182.

234. A construct comprising at least one single variable domain ofaspect 183.

235. A construct comprising at least one single variable domain ofaspect 184.

236. A construct comprising at least one single variable domain ofaspect 185.

237. A construct comprising at least one single variable domain ofaspect 186.

238. A construct comprising at least one single variable domain ofaspect 187.

239. A construct comprising at least one single variable domain ofaspect 188.

240. A construct comprising at least one single variable domain ofaspect 189.

241. A construct comprising at least one single variable domain ofaspect 190.

242. A construct comprising at least one single variable domain ofaspect 191.

243. A construct comprising at least one single variable domain ofaspect 192.

244. A construct comprising at least one single variable domain ofaspect 193.

245. A construct comprising at least one single variable domain ofaspect 194.

246. A construct comprising at least one single variable domain ofaspect 195.

247. A construct comprising at least one single variable domain ofaspect 196.

248. A construct comprising at least one single variable domain ofaspect 197.

249. A construct comprising at least one single variable domain ofaspect 198.

250. A construct comprising at least one single variable domain ofaspect 199.

251. A construct comprising at least one single variable domain ofaspect 200.

252. A construct comprising at least one single variable domain ofaspect 201.

253. A construct comprising at least one single variable domain ofaspect 202.

254. A construct comprising at least one single variable domain ofaspect 203.

255. A construct comprising at least one single variable domain ofaspect 204.

256. A construct comprising at least one single variable domain ofaspect 205.

257. A construct comprising at least one single variable domain ofaspect 206.

258. A construct comprising at least one single variable domain ofaspect 207.

259. A construct comprising at least one single variable domain ofaspect 208.

260. A construct comprising at least one single variable domain ofaspect 209.

261. A construct comprising at least one single variable domain ofaspect 210.

262. A construct comprising at least one single variable domain ofaspect 211.

263. A construct comprising at least one single variable domain ofaspect 212.

264. A construct comprising at least one single variable domain ofaspect 213.

265. A pharmaceutical composition comprising a single variable domain ofaspects 1 to 213 and/or a construct of aspects 214 to 264.

266. A nucleotide sequence encoding for a single variable domain ofaspects 1 to 213 and/or a construct of aspects 214 to 264.

267. A host cell able to express and/or comprising a single variabledomain of aspects 1 to 213 and/or a construct of aspects 214 to 264,and/or a nucleotide sequence of aspect 234.

268. Method of making a single variable domain of any aspects 1 to 213,method of making a construct of aspects 214 to 264; method of making apharmaceutical composition of aspect 265; method of making a nucleotidesequence of aspect 266; and/or making a host cell of 267.

269. Method of screening a molecule specifically binding to a member ofthe Notch pathway using a single variable domain of aspects 1 to 213 ora construct of aspects 214 to 264, a pharmaceutical composition ofaspect 265, a nucleotide sequence of aspect 266, and/or a host cell ofaspects 267.

270. Method for the prevention and/or treatment of at least one diseaseor disorder in which a member selected from the group consisting of theNotch pathway plays a role or is implicated, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of at least one single variable domain of aspects 1 to 213, oneconstruct of aspects 214 to 264, or one pharmaceutical composition ofaspect 265.

271. Method for the prevention and/or treatment of at least one diseaseor disorder selected from the group of diseases consisting of cancers,an autoimmune diseases or neurodegenerative diseases, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of at least one single variable domain ofaspects 1 to 213, one construct of aspects 214 to 264, or onepharmaceutical composition of aspect 265.

272. Use of a pharmaceutically active amount of at least one singlevariable domain of aspects 1 to 213, one construct of aspects 214 to264, or one pharmaceutical composition of aspect 265 for the preventionand/or treatment of at least one disease or disorder in which a memberselected from the group consisting of the Notch pathway plays a role oris implicated.

273. Use of a pharmaceutically active amount of at least one singlevariable domain of aspects 1 to 213, one construct of aspects 214 to264, or one pharmaceutical composition of aspect 265 for the preventionand/or treatment of at least one disease or disorder selected from thegroup of diseases consisting of cancers, an autoimmune diseases orneurodegenerative diseases.

274. Use of a pharmaceutically active amount of at least one singlevariable domain of aspects 1 to 213, one construct of aspects 214 to264, or one pharmaceutical composition of aspect 265 for the manufactureof a medicament for the prevention and/or treatment of at least onedisease or disorder in which a member selected from the group consistingof the Notch pathway plays a role or is implicated.

275. Use of a pharmaceutically active amount of at least one singlevariable domain of aspects 1 to 213, one construct of aspects 214 to264, or one pharmaceutical composition of aspect 265 for the manufactureof a medicament for the prevention and/or treatment of at least onedisease or disorder selected from the group of diseases consisting ofcancers, an autoimmune diseases or neurodegenerative diseases.

1. A single variable domain that specifically binds to at least onemember of the Notch pathway.
 2. The single variable domain according toclaim 1, wherein the member of the Notch pathway is selected from thegroup consisting of the human members of the Notch pathway.
 3. Thesingle variable domain according to claim 1, wherein the member of theNotch pathway is selected from the group consisting of the Notchreceptors and Notch ligands.
 4. The single variable domain according toclaim 1, wherein the member of the Notch pathway is selected from thegroup consisting of the human Notch receptors and human Notch ligands.5. The single variable domain according to claim 1, wherein the memberof the Notch pathway is selected from the group consisting of Jagged-1,Jagged-2, DLL1, DLL3, DLL4, Notch-1, Notch-2, Notch-3, and Notch-4. 6.The single variable domain according to claim 1, wherein the member ofthe Notch pathway is selected from the group consisting of humanJagged-1, human Jagged-2, human DLL1, human DLL3, human DLL4, humanNotch-1, human Notch-2, human Notch-3, and human Notch-4.
 7. The singlevariable domain according to claim 1, wherein the member of the Notchpathway is selected from the group consisting of human Jagged-1, humanDLL4, human Notch-1 and human Notch-2.
 8. The single variable domainaccording to claim 1, wherein the single variable domain additionallyblocks the interaction between at least one member of the Notch ligandswith at least one other member of the Notch receptors.
 9. The singlevariable domain according to claim 1, wherein the single variable domainadditionally blocks the interaction between at least one member of thegroup consisting of Notch ligands and Notch receptors with at least onesingle variable domain with sequences having SEQ ID NO: 329 to
 364. 10.The single variable domain according to claim 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with 80% sequence identity to at least onesequence selected from the group consisting of single variable domainswith sequences having SEQ ID NO: 329 to
 364. 11. The single variabledomain according to claim 1, wherein the single variable domain isselected from the group consisting of a) single variable domains withsequences having SEQ ID NO: 329 to 364; and b) single variable domainswith sequences having SEQ ID NO: 329 to 364, wherein up to 10 amino acidresidues are replaced by naturally occurring amino acids and whereinsaid replaced amino acids are located within the framework regions. 12.The single variable domain according to claim 1, wherein the singlevariable domain is selected from the group consisting of a) singlevariable domains with sequences having SEQ ID NO: 329 to 364; and b)single variable domains with sequences having SEQ ID NO: 329 to 364,wherein up to 8 amino acid residues are replaced by naturally occurringamino acids and wherein said replaced amino acids are located within theframework regions.
 13. The single variable domain according to claim 1,wherein the single variable domain is selected from the group consistingof a) single variable domains with sequences having SEQ ID NO: 329 to364; and b) single variable domains with sequences having SEQ ID NO: 329to 364, wherein up to 5 amino acid residues are replaced by naturallyoccurring amino acids and wherein said replaced amino acids are locatedwithin the framework regions.
 14. The single variable domain accordingto claim 1, wherein the single variable domain is selected from thegroup consisting of a) single variable domains with sequences having SEQID NO: 329 to 364; and b) single variable domains with sequences havingSEQ ID NO: 329 to 364, wherein up to 3 amino acid residues are replacedby naturally occurring amino acids and wherein said replaced amino acidsare located within the framework regions.
 15. The single variable domainaccording to claim 1, wherein the single variable domain is selectedfrom the group consisting of a) single variable domains with sequenceshaving SEQ ID NO: 329 to 364; and b) single variable domains with 80%sequence identity to at least one sequences selected from the groupconsisting of sequences having SEQ ID NO: 329 to 364; and wherein saidselected single variable domain from group a) and b) binds to at leastone member of the Notch signalling pathway with a dissociation constant(K_(D)) of 10⁻⁷ to 10⁻¹² moles/liter or less.